LENTIVIRAL VECTOR BASED IMMUNOLOGICAL COMPOUNDS AGAINST MALARIA

Abstract

The invention relates to lentiviral vector particles pseudotyped with a determined heterologous viral envelope protein or viral envelope proteins originating from a RNA virus and which comprise in its genome at least one recombinant polynucleotide encoding at least one polypeptide(s) carrying epitope(s) of an antigen of a Plasmodium parasite capable of infecting a mammalian host. The lentiviral vector particles are used in order to elicit an immunological response against malaria parasites.

Description

[0001] The invention relates to a lentiviral vector based immunological compounds against malaria.

[0002] In view of the impediments which have been observed in the design of vaccine strategies, many diseases that lead to high mortality and morbidity such as malaria still necessitate the development of new vaccine platforms capable of eliciting strong T-cell mediated immunity and advantageously potent humoral immune responses. Among parasitic infections affecting humans, malaria is a disease for which numerous attempts for such vaccines have been proposed.

[0003] However, concerning malaria, only vaccines containing radiation attenuated sporozoites consistently induce sterile immunity in rodents (Nussenzweig R. S. et al, Nature 216, 160-162 (1967)), monkeys (Gwadz; R. W. et al, Bull World Health Organ 57 Suppl 1, 165-173 (1979)) and humans (Clyde, D. F. et al, Am J. Med Sci 266, 169-177 (1973). Albeit very interesting, the irradiated sporozoite vaccine approach still needs to overcome numerous challenges, in particular related to safety, production, storage and distribution, before it can be considered promising for mass vaccination.

[0004] In a Review Article, Limbach K. J. & Richie T. L. (Parasit immunology, 2009, 31, 501-519) have considered different available vaccine platforms against malaria, which are based on the use of viral vectors as delivery means for the antigens eliciting an immune response. Such platforms include vaccines designed on the basis of poxvirus-vectored malaria antigens, or adenovirus-vectored malaria antigens, both types of vectors being inter alia proposed in approaches involving heterologous prime-boost regimens for administration. Apart from these poxvirus or adenovirus based technologies, the authors of the review disclose that novel vector systems may be promising in animal models considering accordingly yellow fever vectored strategies or alpha virus replicon vectored strategies. They also envisage various potentially interesting ways to overcome the persisting difficulties in designing vaccine vectors that may be efficient in providing long-lasting immunity in mammalian hosts and fulfil the requirements of safety for administration to human hosts. Such ways would include combination of heterologous delivery means for the antigens, use of adjuvants or immunomodulatory components.

[0005] In an attempt to overcome at least some of the drawbacks observed when assaying the proposed vaccine compositions of the prior art, the inventors have considered the approach of lentiviral vectors as a basis for a new vaccination platform with a view to develop prophylactic vaccines against malaria.

[0006] Malaria is a pathology which is transmitted to the host by Anophele mosquitoes and is endemic in many countries where it generates the death of many millions of people each year. Apart from mortality, malaria causes morbidity in a large proportion of the population in countries where it is endemic, thereby causing medical and economic major concerns for these countries.

[0007] Five species of Plasmodium parasites are known to infect human: Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi (17) and Plasmodium falciparum, the latter being the agent of malaria which causes almost all mortality cases. Infection in humans begins with inoculation of sporozoites forms of the parasite that originate from female mosquitoes. These forms of the parasite are rapidly transferred from the blood flow to the liver of the human host where they proceed with invasion of hepatocytes. Depending on the strain of Plasmodium, the duration of the intra-hepatic cycle of the parasite is from 5 to 15 days, the shorter (around 5.5 days) being for P. falciparum. The parasite amplifies in the liver as a result of asexual replication in the infected cells, giving rise to merozoite forms of the parasite. After the merozoites have been liberated from the hepatocytes, they progress toward a blood-stage infection which corresponds to the symptomatic phase of the infection in the human host. Accordingly, the merozoites rapidly penetrate into red blood cells (erythrocytes) through a specific membrane receptor. The merozoite invasion of the erythrocytes corresponds to the erythrocytic stage of the cycle which lasts for 48 to 72 hours depending on the strain of Plasmodium. During this stage, merozoites undergo multiple nuclear divisions, giving rise to the liberation of further merozoites, which are able to perform invasion of additional red blood cells and thus to repeat the cycle. In humans, the symptomatic disease is the result of the effects of invasion of erythrocytes, of their destruction and of the response of the hosts. During the infection, some of the parasital forms differenciate as gametocytes which are then ingested by mosquitoes where they undergo a sporogonic cycle, giving rise to sporozoites which then infect humans.

[0008] Due to the cycle of the infection encompassing distinct stages in the human body, and different forms of the parasite, various strategies have been proposed in order to deliver vaccine principles and various antigens of Plasmodium have been proposed as targets for the immune response, especially when addressing humoral antibody response.

[0009] Target candidates suitable for causing or improving the immune response may encompass various antigens with a view to design a vaccine and accordingly encompass "liver-stage antigens" (also designated as "pre-erythrocytic stage antigens") and/or "blood-stage antigens".

[0010] Within the frame of the present invention, the inventors have primarily considered that pre-erythrocytic stage antigens of malaria parasite may be advantageously used to elicit a protective immune response which would be uniformly effective at the level of a population, because they show less variability than antigens appearing at a later stage in the cycle of the parasite in humans. The inventors have also considered that for a long-term protection against malaria, means suitable to elicit cellular responses would be necessary, and would advantageously be supplemented by humoral responses.

[0011] The inventors have accordingly determined that a protective immune response would require the onset and development of efficient effector cells and memory cells and that said response should be strong enough to outperform the efficiency of the immune response which has been observed to natural infection.

[0012] The invention thus provides a novel lentiviral-based vector, as a new platform for the preparation or development of malaria vaccine which, in addition to the delivery means of the active principle for the immune composition, enables the determination of an appropriate immunization patterns suitable for eliciting a strong and long-lasting immune response when adapted to humans, as proved in a murine model commonly used for malaria.

[0013] The invention accordingly relates to lentiviral vector particles which are lentiviral vector particles, in particular replication-incompetent lentiviral vector particles, especially replication-incompetent HIV-based vector particles characterized in that (i) they are pseudotyped with a determined heterologous viral envelope protein or viral envelope proteins originating from a RNA virus and (ii) they comprise in their genome at least one recombinant polynucleotide encoding at least one polypeptide(s) carrying epitope(s) of a pre-erythrocytic stage antigen of a Plasmodium parasite capable of infecting a mammalian host, wherein said epitope(s) encompass(es) T-epitope(s).

[0014] In a particular embodiment of the invention, the encoded polypeptide of a pre-erythrocytic stage antigen of a Plasmodium parasite further comprises B-epitope(s).

[0015] According to the invention, the lentiviral vector particles are either designed to express proficient (i.e., integrative-competent) or deficient (i.e., integrative-incompetent) particles.

[0016] The expressions "malaria parasite" and "Plasmodium parasite>> are used interchangeably in the present application. They designate every and all forms of the parasite that are associated with the various stages of the cycle in the mammalian, especially human host, including in particular sporozoites, especially spororoites present in the blood flow after inoculation, or sporozoites developing in the hepatocytes, merozoites, including especially merozoites produced in the hepatocytes (forms of the pre-erythrocytic stage and including forms of the erythrocytic stage of the cycle such as merozoites contained in red-blood cells of the cycle). These forms of the parasite are characterized by various specific antigens that are well known and identified in the art, and can also be designated by reference to the stage of the infection.

[0017] The expressions "T-epitope" and "B-epitope" refer to antigenic determinants that are involved respectively in the adaptive immune response driven by T cells and in the immune response driven by B cells. In particular said T-epitopes and respectively B-epitopes elicit T cell, respectively B cell immune response when delivered to the host in suitable conditions.

[0018] The lentiviral vector particles (or lentiviral vectors or lentiviral-based vector particles) defined in the present invention are pseudotyped lentiviral vectors consisting of vector particles bearing envelope protein or envelope proteins which originate from a virus different from the particular lentivirus, especially HIV, in particular HIV-1, which provides the vector genome of the lentiviral vector particles. Accordingly, said envelope protein or envelope proteins, are "heterologous" viral envelope protein or viral envelope proteins with respect to the vector genome of the particles. In the following pages, reference will also be made to "envelope protein(s)" to encompass any type of envelope protein or envelope proteins suitable to perform the invention.

[0019] When reference is made to "lentiviral" vectors (lentiviral-based vectors) in the application, it encompasses, in a particular embodiment, HIV-based vectors and especially HIV-1-based vectors.

[0020] The lentiviral vectors according to the invention are replacement vectors, meaning that the sequences of the original lentivirus encoding the lentiviral proteins are essentially deleted in the genome of the vector or, when present, are modified, and especially mutated, especially truncated, to prevent expression of biologically active lentiviral proteins, in particular, in the case of HIV, to prevent the expression by said transfer vector, of functional ENV, GAG, and POL proteins and optionally of further structural and/or accessory and/or regulatory proteins of the lentivirus, especially of HIV.

[0021] The "vector genome" of the vector particles is a recombinant vector which also comprises the polynucleotide or transgene of interest encoding the polypeptide(s) of malaria parasite. The lentiviral-based sequence and polynucleotide/transgene of the vector genome are borne by a plasmid vector thus giving rise to the "transfer vector" also referred to as "sequence vector". Accordingly, these expressions are used interchangeably in the present application.

[0022] The vector genome as defined herein accordingly contains, apart from the so-called recombinant polynucleotide placed under control of proper regulatory sequences for its expression, the sequences of the original lentiviral genome which are non-coding regions of said genome, and are necessary to provide recognition signals for DNA or RNA synthesis and processing (mini-viral genome). These sequences are cis-acting sequences necessary for packaging, reverse transcription and transcription and furthermore for the particular purpose of the invention, they contain a functional sequence favouring nuclear import in cells and accordingly transgene transfer efficiency in said cells, which element is described as a DNA Flap element and contains or consists of the so-called central cPPT-CTS nucleotidic domain present in lentiviral genome sequences or in some retroelements such as those of yeasts.

[0023] The structure and composition of the vector genome used to prepare the lentiviral vectors of the invention are based on the principles described in the art and on examples of such lentiviral vectors primarily disclosed in (Zennou et al, 2000; Firat H. et al, 2002; VandenDriessche T. et al). Constructs of this type have been deposited at the CNCM (Institut Pasteur, France) as will be referred to herein. In this respect reference is also made to the disclosure, including to the deposited biological material, in patent applications WO 99/55892, WO 01/27300 and WO 01/27304.

[0024] According to a particular embodiment of the invention, a vector genome may be a replacement vector in which all the viral protein coding sequences between the 2 long terminal repeats (LTRs) have been replaced by the recombinant polynucleotide encoding the polypeptide of the malaria parasite, and wherein the DNA-Flap element has been re-inserted in association with the required cis-acting sequences described herein. Further features relating to the composition of the vector genome are disclosed in relation to the preparation of the particles.

[0025] A lentiviral vector particle of the invention may comprise in its genome more than one recombinant polynucleotide encoding at least one polypeptide carrying epitope(s) of a pre-erythrocytic stage antigen as disclosed herein. In particular, said vector genome comprises two polynucleotides which are consecutive or separated on the genome and which encode different polypeptides of either the same or distinct antigens of the pre-erythrocytic stage of a Plasmodium parasite or different polypeptides of distinct antigens of different forms of the malaria parasite, especially antigens of the pre-erythrocytic stage and antigens of the erythrocytic stage of the parasite.

[0026] In a particular embodiment, the vector genome contains two recombinant polynucleotides, each of them encoding a distinct polypeptide and each polypeptide originating from a different antigen of the same stage.

[0027] By the expression "polypeptide carrying epitope(s) of an antigen", it is intended according to the present invention a polypeptide which may be a native antigen of a Plasmodium parasite, a mutated version thereof and in particular a fragment of such a native antigen and especially a truncated version of such a native antigen. A polypeptide has an amino acid sequence which is sufficient to provide one or several epitope(s), and may accordingly have a length of at least about 4 amino acid residues and especially from about 4 to about 8 amino acid residues for conformational B epitopes or at least about 9 amino acid residues and in particular from about 9 to about 19 amino acid residues for sequential T epitopes.

[0028] In a particular embodiment of the invention, the recombinant polynucleotide of the lentiviral vector particles encodes a truncated version of an antigen of the malaria parasite, especially a fragment which results from the deletion of a functional domain of the full-length (i.e., native) antigen, when said domain is not useful or is detrimental to the elicitation of an immune response in a host.

[0029] In a particular embodiment of the invention, the lentiviral vector particles comprise in their genome, at least one recombinant polynucleotide which encodes a polypeptide(s) of an antigen from the circumsporozoite protein of a Plasmodium parasite, esp. of Plasmodium falciparum or Plasmodium malariae, Plasmodium vivax, Plasmodium ovale or Plasmodium knowlesi. It is especially a truncated version of the CSP and in particular a polypeptide devoid of the GPI anchoring motif of the CSP.

[0030] In an embodiment of the invention, the lentiviral vector particles comprise in their genome a recombinant polynucleotide which encodes a polypeptide(s) of an antigen selected from the group of the sporozoite surface protein 2 (TRAP/SSP2), liver-stage antigen (LSA in particular LSA3), Pf exported protein 1 (Pf Exp1)/Py hepatocyte erythrocyte protein (PyHEP17), and Pf antigen 2 (where Pf represents Plasmodium falciparum and Py represents Plamsodium yoelii), sporozoite and liver stage antigen (SALSA), sporozoite threonine and asparagines-rich (STARP) or other pre-erythrocytic antigen, possibly in addition to the polypeptide of an antigen of the CSP.

[0031] In a particular embodiment of the invention, the polypeptide of the antigen of the malaria parasite is a fragment of the CSP protein and it is co-expressed by the vector genome with a polypeptide of another antigen of the malaria parasite, either an antigen from the pre-erythrocytic stage or an antigen of the erythrocytic stage. Antigens of the erythrocytic stage which may be used to design the polynucleotide encoding the polypeptide according to the invention are merozoite surface protein 1 (MSP2), in particular Merozoite surface protein 1 (MSP-1), Merozoite surface protein 2 (MSP-2) merozoite surface protein 3 (MSP-3), Merozoite surface protein 4 (MSP-4), Merozoite surface protein 6 (MSP-6), Ring-infected erythrocyte surface antigen (RESA), Rhoptry associated protein 1 (RAP-1), Apical membrane antigen 1 (AMA-1), Erythrocyte binding antigen (EBA-175), Erythrocyte membrane-associated giant protein or Antigen 332 (Ag332), dnaK-type molecular chaperone, Glutamate-rich protein (GLURP), in particular MSP3-GLURP fusion protein (WO 2004/043488; ref 28), Erythrocyte membrane protein 1 (EMP-1), Serine repeat antigen (SERA), Clustered-asparagine-rich protein (CARP), Cirumsporozoite protein-related antigen precursor (CRA), Cytoadherence-linked asexual protein (CLAG), Acid basic repeat antigen (ABRA) or 101 kDa malaria antigen, Rhoptry antigen protein (RAP-2), Knob-associated histidine-rich protein (KHRP), Rhoptry antigen protein (RAP), Cysteine protease, Hypothetical protein PFE1325w, Protective antigen (MAg-1), Fructose-bisphosphate aldolase, Ribosomal phosphoprotein P0, P-type ATPase, Glucose-regulated protein (GRP78), Asparagine and aspartate-rich protein (AARP1), Interspersed repeat antigen or PFE0070w.}

[0032] Antigens of the sexual stage which may be used to design the polynucleotide encoding the polypeptide according to the invention are Sexual stage and sporozoite surface antigen, Antigen Pfg27/25, Antigen QF122, 11-1 polypeptide, Gametocyte-specific surface protein (Pfs230) Ookinete surface protein (P25), Chitinase, Multidrug resistance protein (MRP).

[0033] These antigens are disclosed by reference to P. falciparum and may have a counterpart in other Plasmodium species. They are reported in Vaughan K. et al (18).

[0034] Vaughan K et al disclose in particular epitopes of said antigens that may be used by the skilled person as a basis to prepare the recombinant polynucleotide(s) used in the vector of the invention.

[0035] The above-cited antigens of Plasmodium parasite have been disclosed in the prior art, including through their sequences which are available in data bases.

[0036] The circumsporozoite protein (CSP) is one of the preferred antigens for the preparation of the lentiviral vector particles of the invention. It constitutes the sporozoite coat protein, which has been recognized in the past as the target of protective antibodies. Apart from its ability to elicit anti-CS antibodies, this antigen further contains T-epitopes including especially CD8+ T-cells epitopes and CD4+ T-cells epitopes. Particular CSP antigens are disclosed through their amino acid sequences as SEQ ID No 20, 23, 26, 27, 28, 29, 30, 31, or as SEQ ID No 32 for a consensus of these sequences. The sequence of P. vivax is given in GenBank as AY674050.1.

[0037] In a particular embodiment of the invention, the lentiviral vector particles have in their genome a recombinant polynucleotide which encodes at least a polypeptide of the CSP-antigen of Plasmodium yoelii as illustrated in the examples or advantageously of Plasmodium falciparum, e.g., a polypeptide corresponding to a fragment of said CSP-antigen devoid of the GPI-anchoring motif in Plasmodium yoelii is CSP DGPI having sequence SEQ ID No 21. Said GPI motif corresponds to the last 12 amino acid residues in the C-terminal part in the native amino acid sequence of the CSP antigen of Plasmodium Yoelii. The counterpart of said fragment of the CSP protein in P. Falciparum is disclosed in the figures and sequences (SEQ ID No 23 for the native protein, SEQ ID No 24 for the sequence devoid of the GPI motif, SEQ ID No 25 for the sequence truncated in the N-terminal end) and used to provide evidence in a suitable murine model, of the capacity of the polypeptide to elicit a protective immune response and even a sterilizing protection against malaria.

[0038] In a particular embodiment of the invention, polynucleotide(s) of the lentiviral vector particles has(have) a mammalian codon optimized (CO) nucleotide sequence and optionally the lentiviral sequences of the genome of said particles has a mammalian codon optimized nucleotide sequence.

[0039] It has been observed that codon optimized nucleotide sequences, especially when optimized for expression in mammalian and in particular in human cells, enable the production of higher yield of particles in such mammalian or human cells. Production cells are illustrated in the examples. Accordingly, when lentiviral vector particles of the invention are administered to a mammalian, especially to a human host, higher amounts of particles are produced in said host which favour the elicitation of a strong immune response.

[0040] In a particular embodiment of the invention, the lentiviral vector particles disclosed herein further contain in their genome, a recombinant polynucleotide which encodes a polypeptide of an antigen of the blood stage of the cycle of the parasite as disclosed above and/or an antigen of the sexual stage.

[0041] The polypeptide is either the native antigen or a modified version thereof, especially a fragment which comprises or consists in T-cell epitope(s) or B-cell epitope(s) or both.

[0042] Examples of polypeptides expressed as a result of administering the lentiviral vector particles of the invention, are the polypeptides encoded by the vector plasmids (or sequence vectors) disclosed hereafter.

[0043] The invention also relates especially to these vector plasm ids, deposited at the CNCM (Paris, France) on Apr. 20, 2010 and having the following features and accession number

TABLE-US-00001 pTRIP-|delta|U3-CMV-MSP1₄₂-CO-WPRE CNCM I-4303 pTRIP-|delta|U3-CMV-Hep17-CO-WPRE CNCM I-4304 pTRIP-|delta|U3-CMV-Hep17|delta|SP-CO-WPRE CNCM I-4305 pTRIP-|delta|U3-CMV-CSP-CO-WPRE CNCM I-4306 pTRIP-|delta|U3-CMV-CSP|delta|SP-CO-WPRE CNCM I-4307 pTRIP-|delta|U3-CMV-CSP|delta|GPI-CO-WPRE CNCM I-4308 pTRIP-|delta|U3-CMV-CSP|delta|SP|delta|GPI-CO-WPRE CNCM I-4309

[0044] These plasmids are described in the figures and sequences of the present application. The sequence of the transgene that they contain is from P. yoelii. Said transgene may be advantageously replaced by the appropriate sequence from P. Falciparum.

[0045] The invention also concerns variants of these plasmids, where the polynucleotide encoding the polynucleotide of a Plasmodium antigen is modified to encode a functional immunogenic variant thereof or is substituted by a corresponding polynucleotide codon optimized from another Plasmodium strain especially from Plasmodium falciparum is modified to substitute the CMV promoter by one of the herein cited promoters.

[0046] In the deposited plasmids the polynucleotide encoding the polypeptide of a Palsmodium yoelii antigen is codon optimized.

[0047] According to the invention, the lentiviral vector particles are pseudotyped with a heterologous viral envelope protein or viral polyprotein of envelope originating from a RNA virus which is not the lentivirus providing the lentiviral sequences of the genome of the lentiviral particles.

[0048] As examples of typing envelope proteins for the preparation of the lentiviral vector particles, the invention relates to viral transmembrane glycosylated (so-called G proteins) envelope protein(s) of a Vesicular Stomatitis Virus (VSV), which is(are) for example chosen in the group of VSV-G protein(s) of the Indiana strain, VSV-G protein(s) of the New Jersey strain, VSV-G protein(s) of the Cocal strain, VSV-G protein of the Isfahan strain, VSV-G protein(s) of Chandipura strain, VSV-G protein(s) of Pyri strain or VSV-G protein(s) of the SVCV strain.

[0049] The envelope glycoprotein of the vesicular stomatitis virus (VSV-G) is a transmembrane protein that functions as the surface coat of the wild type viral particles. It is also a suitable coat protein for engineered lentiviral vectors. Presently, nine virus species are definitively classified in the VSV gender, and nineteen rhabdoviruses are provisionally classified in this gender, all showing various degrees of cross-neutralisation. When sequenced, the protein G genes indicate sequence similarities. The VSV-G protein presents a N-terminal ectodomain, a transmembrane region and a C-terminal cytoplasmic tail. It is exported to the cell surface via the transGolgi network (endoplasmic reticulum and Golgi apparatus).

[0050] Vesicular stomatitis Indiana virus (VSIV) and Vesicular stomatitis New Jersey virus (VSNJV) are preferred strains to pseudotype the lentiviral vectors of the invention, or to design recombinant envelope protein(s) to pseudotype the lentiviral vectors. Their VSV-G proteins are disclosed in GenBank, where several strains are presented. For VSV-G New Jersey strain reference is especially made to the sequence having accession number V01214. For VSV-G of the Indiana strain, reference is made to the sequence having accession number AAA48370.1 in Genbank corresponding to strain J02428.

[0051] Alternatively, among VSV, Chandipura virus (CHPV), Cocal virus (COCV), Perinet virus (PERV), Piry virus (PIRYV), SVCV or Isfahan virus may be good candidates to design pseudotyping envelope proteins, and especially to prepare particles used for a boosting step of the immunization, accordingly providing second envelope protein(s) or third envelope protein(s), or further envelope protein(s) when the vector particles of the invention are used in a prime-boost administration regimen. When used accordingly, Cocal virus envelope protein(s) would be preferred for a late or last administration in a prime-boost regimen. However, Chandipura virus (CHPV) and Piry virus (PIRYV) may provide envelope proteins having low fusogenicity as a result of their lower affinity for their receptor, when comparing the vector titers obtained with particles prepared with different envelopes. Therefore in a first approach these envelopes may be excluded from the choice of envelopes in order to prepare particles with an efficient transduction capacity.

[0052] The amino acid sequences and coding sequences of the VSV-G proteins referred to herein are disclosed in patent application WO 2009/019612. Particular examples of these amino acid sequences are also provided in the present application as SEQ ID No 77, 79, 82, 84, 86, 88, 90. Plasmids containing VSV-G encoding sequences are described in said application WO 2009/019612 which is incorporated by reference. The plasmids have been deposited at the CNCM (Paris, France). Nucleotide sequences encoding said envelope proteins are disclosed in the present application as SEQ ID No 76, 78, 81, 83, 85, 87, 89.

[0053] In a particular embodiment of the invention, said first and second and if any said third or further, viral envelope protein(s) are capable of uptake by antigen presenting cells and especially by dendritic cells including by liver dendritic cells by mean of fusion and/or of endocytosis. In a particular embodiment, the efficiency of the uptake may be used as a feature to choose the envelope of a VSV for pseudotyping. In this respect the relative titer of transduction (Titer DC/Titer of other transduced cells e.g. 293T cells) may be considered as a test and envelope having a relative good ability to fuse with DC would be preferred. Relative titers of transduction are illustrated in the examples.

[0054] Antigen Presenting Cells (APC) and especially Dentritic cells (DC) are proper target cells for pseudotyped lentiviral vectors which are used as immune compositions accordingly.

[0055] Polynucleotide encoding VSV envelope protein(s) (VSV-G) also targets splenocytes, in particular Antigen Presenting Cells (APC) or Dendritic Cells (DC), or liver cells including liver dendritic cells, hepatocytes or non parenchymal cells.

[0056] The envelope protein(s), also designated sometimes as surface protein in particular viruses, are said to "originate" from a different organism, and especially from different RNA virus strains, meaning that in said protein(s), essential features of the corresponding protein(s) expressed in a determined RNA virus are maintained. Said essential features, relate to the structure or to the function of the protein and are those which enable especially the obtained protein(s) to be expressed at the surface of the vector particles for pseudotyping said vectors. The envelope proteins are then capable of being recognized and internalized in the target cells of the hosts when present on the vector particles.

[0057] In a particular embodiment, protein(s) or glycoprotein(s), suitable for use in the design of pseudotyped lentiviral vectors of the kit of compounds are used as multimeric proteins, such as VSV-G protein which is trimeric.

[0058] The envelope protein(s) are expressed from a polynucleotide containing the coding sequence for said protein(s), which polynucleotide is inserted in a plasmid (designated envelope expression plasmid or pseudotyping env plasmid) used for the preparation of the lentiviral vector particles of the invention. The polynucleotide encoding the envelope protein(s) is under the control of regulatory sequences for the transcription and/or expression of the coding sequence (including optionally post-transcriptional regulatory elements (PRE) especially a polynucleotide such as the element of the Woodchuck hepatitis virus, i.e. the WPRE sequence obtainable from Invitrogen).

[0059] Accordingly, a nucleic acid construct is provided which comprises an internal promoter suitable for the use in mammalian cells, especially in human cells in vivo and the nucleic acid encoding the envelope protein under the control of said promoter. A plasmid containing this construct is used for transfection or for transduction of cells suitable for the preparation of particles. Promoters may in particular be selected for their properties as constitutive promoters, tissue-specific promoters, or inducible promoters. Examples of suitable promoters encompass the promoters of the following genes: EF1α, human PGK, PPI (preproinsulin), thiodextrin, HLA DR invariant chain (P33), HLA DR alpha chain, Ferritin L chain or Ferritin H chain, Chymosin beta 4, Chymosin beta 10, Cystatin Ribosomal Protein L41, CMVie or chimeric promoters such as GAG(CMV early enhancer/chicken β actin) disclosed in Jones S. et al (19).

[0060] These promoters may also be used in regulatory expression sequences involved in the expression of gag-pol derived proteins from the encapsidation plasmids.

[0061] Alternatively, when the envelope expression plasmid is intended for expression in stable packaging cell lines, especially for stable expression as continuously expressed viral particles, the internal promoter to express the envelope protein(s) is advantageously an inducible promoter such as one disclosed in Cockrell A. S. et al. (20). As examples of such promoters, reference is made to tetracycline and ecdysone inducible promoters. The packaging cell line may be the STAR packaging cell line (ref 20, 21) or a SODk packaging cell line, such as SODk0 derived cell lines, including SODk1 and SODk3 (ref 20, 22, 23, 24).

[0062] The nucleotide sequence used for the expression of the envelope protein(s) required for pseudotyping the lentiviral vector particles may alternatively be modified, thus providing a variant with respect to the nucleic acid encoding the native envelope protein(s) used as reference. The modification may be carried out to improve the codons usage (codon optimization) in the cells for the preparation of the vector particles and/or in the transduced cells of the host. It may be modified to express a protein different from the native protein(s), especially one which has an improved pseudotyping capacity, an improved capacity in the level of production, or an improved capacity with respect to prevention of sero-neutralization (also designated as cross-reactive proteins) with other envelope protein(s) used in the kit of compounds.

[0063] Such a modification of the polynucleotide encoding the envelope proteins(s) or modification of the envelope protein(s) (to generate variants of native envelopes) may affect and especially improve their level of production in a cell host or their ability to pseudotype the vector particles possibly by improving the density of envelope protein(s) associated with pseudovirions. Said modification may derive from a mutation in the amino acid sequence of said protein(s), for instance by addition, deletion or substitution of one or several nucleotides or nucleotidic fragments or may relate to post translational modifications and in particular to the glycosylation status of said envelope protein(s).

[0064] The envelope protein(s) used to pseudotype the lentiviral vectors of the invention are indeed especially glycoproteins.

[0065] It has already been shown that pseudotyping viral vectors with Vesicular Stomatitis Virus glycoprotein (VSV-G) enables the transduction of a large range of cell types from different species. This VSV-G glycoprotein, in addition to its broad tropism, has an interesting stability when used for vector pseudotyping. Therefore, VSV-G have been used as a standard for evaluating the efficiency of other pseudotypes (Cronin J. et al, 2005).

[0066] According to the invention, the lentiviral vector particles are the product recovered from co-transfection of mammalian cells, with:

[0067] a vector plasmid comprising (i) lentiviral, especially HIV-1, cis-active sequences necessary for packaging, reverse transcription, and transcription and further comprising a functional lentiviral, especially derived from HIV-1, DNA flap element and (ii) a polynucleotide encoding a polypeptide of an antigen of a malaria parasite as disclosed herein under the control of regulatory expression sequences, and optionally comprising sequences for integration;

[0068] an expression plasmid encoding a pseudotyping envelope derived from a RNA virus, said expression plasmid comprising a polynucleotide encoding an envelope protein or proteins for pseudotyping, wherein said envelope pseudotyping protein is advantageously from a VSV and is in particular a VSV-G or a variant thereof and,

[0069] an encapsidation plasmid, which either comprises lentiviral, especially HIV-1, gag-pol packaging sequences suitable for the production of integration-competent vector particles or modified gag-pol packaging sequences suitable for the production of integration-deficient vector particles.

[0070] The invention thus also concerns lentiviral vector particles as described above, which are the product recovered from a stable cell line with

[0071] a vector plasmid comprising (i) lentiviral, especially HIV-1, cis-active sequences necessary for packaging, reverse transcription, and transcription and further comprising a functional lentiviral, especially HIV-1, DNA flap element and optionally comprising cis-active sequences necessary for integration, said vector plasmid further comprising (ii) a polynucleotide of a truncated mammalian, especially human, codon-optimized sequence of the cs gene of a Plasmodium parasite, under the control of regulatory expression sequences, especially a promoter;

[0072] a VSV-G envelope expression plasmid comprising a polynucleotide encoding a VSV-G envelope protein or envelope proteins, wherein said polynucleotide is under the control of regulating expression sequences, in particular regulatory expression sequences comprising an inducible promoter, and;

[0073] an encapsidation plasmid, wherein the encapsidation plasmid either comprises lentiviral, especially HIV-1, gag-pol coding sequences suitable for the production of integration-competent vector particles or modified gag-pol coding sequences suitable for the production of integration-deficient vector particles, wherein said gag-pol sequences are from the same lentivirus sub-family as the DNA flap element, wherein said lentiviral gag-pol or modified gag-pol sequence is under the control of regulating expression sequences.

[0074] The stable cell lines expressing the vector particles of the invention are in particular obtained by transduction of the plasm ids.

[0075] The polynucleotide encodes at least one polypeptide of a malaria antigen according to any embodiment disclosed in the present application. In particular, it encodes a polypeptide which is a truncated mammalian, especially human, codon-optimized sequence of the cs gene of a Plasmodium parasite, especially of Plasmodium falciparum.

[0076] In a particular embodiment, the polynucleotide encodes another polypeptide of a distinct antigen of the malaria parasite, or it encodes two or more polypeptides which originate and/or are derived from distinct antigens of said parasite as disclosed in the various embodiments. Accordingly, the vector plasmid may comprise several expression cassettes for the expression of the various polypeptides or may comprise bicistronic or multicistronic expression cassettes where the polynucleotides encoding the various polypeptides are separated by an IRES sequence of viral origin (Internal Ribosome Entry Site), or it may encode fusion protein(s).

[0077] The internal promoter contained the vector genome and controlling the expression of the polynucleotide encoding a polypeptide of an antigen of the malaria parasite (as a transgene or in an expression cassette) may be selected from the promoters of the following genes: EF1α, human PGK, PPI (preproinsulin), thiodextrin, HLA DR invariant chain (P33), HLA DR alpha chain, Ferritin L chain or Ferritin H chain, Chymosin beta 4, Chimosin beta 10, or Cystatin Ribosomal Protein L41 CMVie or chimeric promoters such as GAG(CMV early enhancer/chicken β actin) disclosed in Jones S. et al (19).

[0078] A promoter among the above cited internal promoters may also be selected for the expression of the envelope protein(s) and packaging (gag-pol derived) proteins.

[0079] Alternatively, vector particles can be produced from co-transfection of the plasmids disclosed herein, in stable packaging cell lines which thus become capable of continuously secreting vector particles. Promoters used in the regulatory expression sequences involved for the expression of the envelope protein(s) are advantageously inducible promoters.

[0080] The following particular embodiments may be carried out when preparing the lentiviral vector particles based on human lentivirus, and especially based on HIV virus.

[0081] According to the invention, the genome of the lentiviral vector particles is derived from a human lentivirus, especially from the HIV lentivirus. In particular, the pseudotyped lentiviral vector is an HIV-based vector, such as an HIV-1, or HIV-2 based vector, in particular is derived from HIV-1M, for example from the BRU or LAI isolates. Alternatively, the lentiviral vector providing the necessary sequences for the vector genome may be originating from lentiviruses such as EIAV, CAEV, VISNA, FIV, BIV, SIV, HIV-2, HIV-O which are capable of transfecting human cells.

[0082] As stated above, when considering it apart from the recombinant polynucleotide that it finally contains, the vector genome is a replacement vector in which the nucleic acid between the 2 long terminal repeats (LTRs) in the original lentivirus genome have been restricted to cis-acting sequences for DNA or RNA synthesis and processing, including for the efficient delivery of the transgene to the nuclear of cells in the host, or at least are deleted or mutated for essential nucleic acid segments that would enable the expression of lentiviral structure proteins including biological functional GAG polyprotein and possibly POL and ENV proteins.

[0083] In a particular embodiment, the vector genome is defective for the expression of biologically functional GAG, and advantageously for biologically functional POL and ENV proteins. Accordingly, the vector genome is devoid of the sequence encoding these proteins.

[0084] In a particular embodiment, the 5' LTR and 3' LTR sequences of the lentivirus are used in the vector genome, but the 3'-LTR at least is modified with respect to the 3'LTR of the original lentivirus at least in the U3 region which for example can be deleted or partially deleted for the enhancer. The 5'LTR may also be modified, especially in its promoter region where for example a Tat-independent promoter may be substituted for the U3 endogenous promoter.

[0085] In a particular embodiment the vector genome comprises one or several of the coding sequences for Vif-, Vpr, Vpu- and Nef-accessory genes (for HIV-1 lentiviral vectors). Alternatively, these sequences can be deleted independently or each other or can be non-functional.

[0086] The vector genome of the lentiviral vector particles comprises, as an inserted cis-acting fragment, at least one polynucleotide consisting in the DNA flap element or containing such DNA flap element. In a particular embodiment, the DNA flap is inserted upstream of the polynucleotide encoding the polypeptide of a malaria antigen, and is advantageously--although not necessarily--located in an approximate central position in the vector genome. A DNA flap suitable for the invention may be obtained from a retrovirus, especially from a lentivirus, in particular a human lentivirus especially a HIV-1 retrovirus, or from a retrovirus-like organism such as retrotransposon. It may be alternatively obtained from the CAEV (Caprine Arthritis Encephalitis Virus) virus, the EIAV (Equine Infectious Anaemia Virus) virus, the VISNA virus, the SIV (Simian Immunodeficiency Virus) virus or the FIV (Feline Immunodeficiency Virus) virus. The DNA flap may be either prepared synthetically (chemical synthesis) or by amplification of the DNA providing the DNA Flap from the appropriate source as defined above such as by Polymerase chain reaction (PCR). In a more preferred embodiment, the DNA flap is obtained from an HIV retrovirus, for example HIV-1 or HIV-2 virus including any isolate of these two types.

[0087] The DNA flap (defined in Zennou V. et al. ref 27, 2000, Cell vol 101, 173-185 or in WO 99/55892 and WO 01/27304), is a structure which is central in the genome of some lentiviruses especially in HIV, where it gives rise to a 3-stranded DNA structure normally synthesized during especially HIV reverse transcription and which acts as a cis-determinant of HIV genome nuclear import. The DNA flap enables a central strand displacement event controlled in cis by the central polypurine tract (cPPT) and the central termination sequence (CTS) during reverse transcription. When inserted in lentiviral-derived vectors, the polynucleotide enabling the DNA flap to be produced during reverse-transcription, stimulates gene transfer efficiency and complements the level of nuclear import to wild-type levels (Zennou et al., Cell, 2000).

[0088] Sequences of DNA flaps have been disclosed in the prior art, especially in the above cited patent applications. These sequences are also disclosed as SEQ ID NO 69 to SEQ ID NO 75. They are preferably inserted as a fragment, optionally with additional flanking sequences, in the vector genome, in a position which is preferably near the centre of said vector genome. Alternatively they may be inserted immediately upstream from the promoter controlling the expression of the polynucleotide(s) of the invention. Said fragments comprising the DNA flap, inserted in the vector genome may have a sequence of about 80 to about 200 bp, depending on its origin and preparation.

[0089] According to a particular embodiment, a DNA flap has a nucleotide sequence of about 90 to about 140 nucleotides.

[0090] In HIV-1, the DNA flap is a stable 99-nucleotide-long plus strand overlap. When used in the genome vector of the lentiviral vector of the invention, it may be inserted as a longer sequence, especially when it is prepared as a PCR fragment. A particular appropriate polynucleotide comprising the structure providing the DNA flap is a 178-base pair polymerase chain reaction (PCR) fragment encompassing the cPPT and CTS regions of the HIV-1 DNA (Zennou et al 2000).

[0091] This PCR fragment may especially be derived from infective DNA clone of HIV-1 LAI especially pLAI3 of HIV1, as a fragment corresponding to the sequence from nucleotide 4793 to 4971. If appropriate, restriction sites are added to one or both extremities of the obtained fragment, for cloning. For example, Nar I restriction sites may be added to the 5' extremities of primers used to perform the PCR reaction.

[0092] Therefore, the DNA flap used in the present invention, is deleted from the unnecessary 5' and 3' parts of the pol gene of the original lentiviral genome and is recombined with sequences of different origin.

[0093] It is specified that the DNA flap used in the genome vector and the polynucleotides of the encapsidation plasmid encoding the GAG and POL polyproteins should originate from the same lentivirus sub-family or from the same retrovirus-like organism.

[0094] Preferably, the other cis-activating sequences of the genome vector also originate from the same lentivirus or retrovirus-like organism, as the one providing the DNA flap.

[0095] The vector genome may further comprise one or several unique restriction site(s) for cloning the recombinant polynucleotide.

[0096] In a preferred embodiment, in said vector genome, the 3' LTR sequence of the lentiviral vector genome is devoid of at least the activator (enhancer) and possibly the promoter of the U3 region. In another particular embodiment, the 3' LTR region is devoid of the U3 region (delta U3). In this respect, reference is made to the description in WO 01/27300 and WO 01/27304.

[0097] In a particular embodiment, in the vector genome, the U3 region of the LTR 5' is replaced by a non lentiviral U3 or by a promoter suitable to drive tat-independent primary transcription. In such a case, the vector is independent of tat transactivator.

[0098] The vector genome also comprises the psi (ψ) packaging signal. The packaging signal is derived from the N-terminal fragment of the gag ORF. In a particular embodiment, its sequence could be modified by frameshift mutation(s) in order to prevent any interference of a possible transcription/translation of gag peptide, with that of the transgene.

[0099] The vector genome may optionally also comprise elements selected among a splice donor site (SD), a splice acceptor site (SA) and/or a Rev-responsive element (RRE).

[0100] According to a particular embodiment, the vector plasmid (or added genome vector) comprises the following cis-acting sequences for a transgenic expression cassette:

[0101] 1. The LTR sequence (Long-Terminal Repeat), required for reverse transcription, the sequences required for transcription and including optionally sequences for viral DNA integration. The 3' LTR is deleted in the U3 region at least for the promoter to provide SIN vectors (Self-inactivating), without perturbing the functions necessary for gene transfer, for two major reasons: first, to avoid trans-activation of a host gene, once the DNA is integrated in the genome and secondly to allow self-inactivation of the viral cis-sequences after retrotranscription. Optionally, the tat-dependent U3 sequence from the 5'-LTR which drives transcription of the genome is replaced by a non endogenous promoter sequence. Thus, in target cells only sequences from the internal promoter will be transcribed (transgene).

[0102] 2. The ψ region, necessary for viral RNA encapsidation.

[0103] 3. The RRE sequence (REV Responsive Element) allowing export of viral messenger RNA from the nucleus to the cytosol after binding of the Rev protein.

[0104] 4. The DNA flap element (cPPT/CTS, normally contained in Pol) to facilitate nuclear import.

[0105] 5. Optionally post-transcriptional elements such as the WPRE cis-active sequence (Woodchuck hepatitis B virus Post-Responsive Element) also added to optimize stability of mRNA (Zufferey et al., 1999), the matrix or scaffold attachment regions (SAR and MAR sequences) such as those of the immunoglobulin-kappa gene (Park F. et al Mol Ther 2001; 4: 164-173).

[0106] The lentiviral vector of the invention is non replicative (replication-incompetent) i.e., the vector and lentiviral vector genome are regarded as suitable to alleviate concerns regarding replication competent lentiviruses and especially are not able to form new particles budding from the infected host cell after administration. This may be achieved in well known ways as the result of the absence in the lentiviral genome of the gag, pol or env genes, or their absence as "functional genes". The gag and pol genes are thus, only provided in trans. This can also be achieved by deleting other viral coding sequence(s) and/or cis-acting genetic elements needed for particles formation.

[0107] By "functional" it is meant a gene that is correctly transcribed, and/or correctly expressed. Thus, if present in the lentiviral vector genome of the invention in this embodiment contains sequences of the gag, pol, or env are individually either not transcribed or incompletely transcribed; the expression "incompletely transcribed" refers to the alteration in the transcripts gag, gag-pro or gag-pro-pol, one of these or several of these being not transcribed. Other sequences involved in lentiviral replication may also be mutated in the vector genome, in order to achieve this status. The absence of replication of the lentiviral vector should be distinguished from the replication of the lentiviral genome. Indeed, as described before, the lentiviral genome may contain an origin of replication ensuring the replication of the lentiviral vector genome without ensuring necessarily the replication of the vector particles.

[0108] In order to obtain lentiviral vectors according to the invention, the vector genome (as a vector plasmid) must be encapsidated in particles or pseudo-particles. Accordingly, lentiviral proteins, except the envelope proteins, have to be provided in trans to the vector genome in the producing system, especially in producing cells, together with the vector genome, having recourse to at least one encapsidation plasmid carrying the gag gene and either the pol lentiviral gene or an integrative-incompetent pol gene, and preferably lacking some or all of the coding sequences for Vif-, Vpr, Vpu- and Nef-accessory genes (for HIV-1 lentiviral vectors).

[0109] A further plasmid is used, which carries a polynucleotide encoding the envelope pseudotyping protein(s) selected for pseudotyping lentiviral vector particles.

[0110] In a preferred embodiment, the packaging plasmid encodes only the lentiviral proteins essential for viral particle synthesis. Accessory genes whose presence in the plasmid could raise safety concerns are accordingly removed. Accordingly, viral proteins brought in trans for packaging are respectively as illustrated for those originating from HIV-1:

[0111] 1. GAG proteins for building of the matrix (MA, with apparent Molecular Weight p17), the capsid (CA, p24) and nucleocapsid (NC, p6).

[0112] 2. POL encoded enzymes: integrase, protease and reverse transcriptase.

[0113] 3. TAT and REV regulatory proteins, when TAT is necessary for the initiation of LTR-mediated transcription; TAT expression may be omitted if the U3 region of 5'LTR is substituted for a promoter driving tat-independent transcription. REV may be modified and accordingly used for example in a recombinant protein which would enable recognition of a domain replacing the RRE sequence in the vector genome, or used as a fragment enabling binding to the RRE sequence through its RBD (RNA Binding Domain).

[0114] In order to avoid any packaging of the mRNA generated from the genes contained in the packaging plasmid in the viral particles, the ψ region is removed from the packaging plasmid. A heterologous promoter is inserted in the plasmid to avoid recombination issues and a poly-A tail is added 3' from the sequences encoding the proteins. Appropriate promoters have been disclosed above.

[0115] The envelope plasmid encodes the envelope protein(s) for pseudotyping which are disclosed herein, under the control of an internal promoter, as disclosed herein.

[0116] Any or all the described plasmids for the preparation of the lentiviral vector particles of the invention may be codon optimized (CO) in the segment encoding proteins. Codon optimization according to the invention is preferably performed to improve translation of the coding sequences contained in the plasmids, in mammalian cells, especially human cells. According to the invention, codon optimization is especially suited to directly or indirectly improve the preparation of the vector particles or to improve their uptake by the cells of the host to whom they are administered, or to improve the efficiency of the transfer of the polynucleotide encoding the polypeptide of an antigen of the malaria parasite (transgene) in the genome of the transduced cells of the host. Methods for optimizing codons are well known in the art and codon optimization is especially performed using available programs to that effect. Codon optimization is illustrated for the coding sequences contained in the described pTRIP or pThV plasmids of the invention illustrated in the examples.

[0117] In a particular embodiment of the invention, the pseudotyped lentiviral vector is also, or alternatively, integrative-incompetent. In such a case, the vector genome and thus the recombinant polynucleotide which it contains do not integrate into the genome of the transduced cells or in the cells of the host to whom it has been administered.

[0118] The present invention relates to the use of a lentiviral vector wherein the expressed integrase protein is defective and which further comprises a polynucleotide especially encoding at least one polypeptide carrying epitope(s) of a pre-erythrocytic stage antigen of a Plasmodium parasite, in an immunogenic composition.

[0119] By "integration-incompetent", it is meant that the integrase, preferably of lentiviral origin, is devoid of the capacity of integration of the lentiviral genome into the genome of the host cells i.e., an integrase protein mutated to specifically alter its integrase activity.

[0120] Integration-incompetent lentiviral vectors are obtained by modifying the pol gene encoding the Integrase, resulting in a mutated pol gene encoding an integrative deficient integrase, said modified pol gene being contained in the encapsidation plasmid. Such integration-incompetent lentiviral vectors have been described in patent application WO 2006/010834. Accordingly the integrase capacity of the protein is altered whereas the correct expression from the encapsidation plasmid of the GAG, PRO and POL proteins and/or the formation of the capsid and hence of the vector particles, as well as other steps of the viral cycle, preceding or subsequent to the integration step, such as the reverse transcription, the nuclear import, stay intact. An integrase is said defective when the integration that it should enable is altered in a way that an integration step takes place less than 1 over 1000, preferably less than 1 over 10000, when compared to a lentiviral vector containing a corresponding wild-type integrase.

[0121] In a particular embodiment of the invention, the defective integrase results from a mutation of class 1, preferably amino acid substitutions (one-amino acid substitution) or short deletions fulfilling the requirements of the expression of a defective integrase. The mutation is carried out within the pol gene. These vectors may carry a defective integrase with the mutation D64V in the catalytic domain of the enzyme, which specifically blocks the DNA cleaving and joining reactions of the integration step. The D64V mutation decreases integration of pseudotyped HIV-1 up to 1/10,000 of wild type, but keep their ability to transduce non dividing cells, allowing efficient transgene expression.

[0122] Other mutations in the pol gene which are suitable to affect the integrase capacity of the integrase of HIV-1 are the following: H12N, H12C, H16C, H16V, S81R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D116I, D116A, N120G, N120I, N120E, E152G, E152A, D-35-E, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H.

[0123] In a particular embodiment, mutation in the pol gene is performed at either of the following positions D64, D116 or E152, or at several of these positions which are in the catalytic site of the protein. Any substitution at these positions is suitable, including those described above.

[0124] Another proposed substitution is the replacement of the amino acids residues RRK (positions 262 to 264) by the amino acids residues AAH.

[0125] In a particular embodiment of the invention, when the lentiviral vector is integration-incompetent, the lentiviral genome further comprises an origin of replication (ori), whose sequence is dependent on the nature of cells where the lentiviral genome has to be expressed. Said origin of replication may be from eukaryotic origin, preferably of mammalian origin, most preferably of human origin. It may alternatively be of viral origin, especially coming from DNA circular episomic viruses, such as SV40 or RPS. It is an advantageous embodiment of the invention to have an origin or replication inserted in the lentiviral genome of the lentiviral vector of the invention. Indeed, when the lentiviral genome does not integrate into the cell host genome (because of the defective integrase), the lentiviral genome is lost in cells that undergo frequent cell divisions; this is particularly the case in immune cells, such as B or T cells. The presence of an origin of replication ensures that at least one lentiviral genome is present in each cell, even after cell division, accordingly maximazing the efficiency of the immune response.

[0126] The lentiviral vector genome of said lentiviral vectors of the invention may especially be derived from HIV-1 plasmid pTRIPΔU3.CMV-GFP deposited at the CNCM (Paris, France) on Oct. 11, 1999 under number 1-2330 (also described in WO01/27300). The sequence of pTRIPΔU3.CMV-eGFP is provided as SEQ ID No 35 and is described in FIG. 11.

[0127] When the vector genome is derived from these particular plasmids, a sequence of a recombinant polynucleotide as disclosed in the present application is inserted therein, in addition or in replacement of the GFP coding fragment. The GFP coding sequence may also be substituted by a different marker. The CMV promoter may also be substituted by another promoter, especially one of the promoters disclosed above, especially in relation to the expression of the transgene.

[0128] The WPRE sequence also contained in the particular deposited pTRIP vectors may optionally be deleted.

[0129] Vector particles may be produced after transfection of appropriate cells (such as mammalian cells or human cells, such as Human Embryonic Kidney cells illustrated by 293 T cells) by said plasmids, or by other processes. In the cells used for the expression of the lentiviral particles, all or some of the plasmids may be used to stably express their coding polynucleotides, or to transiently or semi-stably express their coding polynucleotides.

[0130] The concentration of particles produced can be determined by measuring the P24 (capsid protein for HIV-1) content of cell supernatants.

[0131] The lentiviral vector of the invention, once administered into the host, infects cells of the host, possibly specific cells, depending on the envelope proteins it was pseudotyped with. The infection leads to the release of the lentiviral vector genome into the cytoplasm of the host cell where the retrotranscription takes place. Once under a triplex form (via the DNA flap), the lentiviral vector genome is imported into the nucleus, where the polynucleotide(s) encoding polypeptide(s) of antigen(s) of the malaria parasite is (are) expressed via the cellular machinery. When non-dividing cells are transduced (such as DC), the expression may be stable. When dividing cells are transduced, such as B cells, the expression is temporary in absence of origin of replication in the lentiviral genome, because of nucleic acid dilution and cell division. The expression may be longer by providing an origin of replication ensuring a proper diffusion of the lentiviral vector genome into daughter cells after cell division. The stability and/or expression may also be increased by insertion of MAR (Matrix Associated Region) or SAR (Scaffold Associated Region) elements in the vector genome.

[0132] Indeed, these SAR or MAR regions are AT-rich sequences and enable to anchor the lentiviral genome to the matrix of the cell chromosome, thus regulating the transcription of the polynucleotide encoding at least one antigenic polypeptide, and particularly stimulating gene expression of the transgene and improving chromatin accessibility.

[0133] If the lentiviral genome is non integrative, it does not integrate into the host cell genome. Nevertheless, the at least one polypeptide encoded by the transgene is sufficiently expressed and longer enough to be processed, associated with MHC molecules and finally directed towards the cell surface. Depending on the nature of the polynucleotide(s) encoding polypeptide(s) of antigen(s) of malaria parasite, the at least one polypeptide epitope associated with the MHC molecule triggers a humoral or a cellular immune response.

[0134] Unless otherwise stated, or unless technically not relevant, the characteristics disclosed in the present application with respect to any of the various features, embodiments or examples of the structure or use of the lentiviral particles, especially regarding their envelope protein(s), or the recombinant polynucleotide, may be combined according to any possible combinations.

[0135] The invention further relates to a combination of compounds for separate administration to a mammalian host, which comprises at least:

[0136] (i) lentiviral vector particles of the invention which are pseudotyped with a first determined heterologous viral envelope pseudotyping protein or viral envelope pseudotyping proteins;

[0137] (ii) provided separately from lentiviral vector particles in (i), lentiviral vector particles of the invention which are pseudotyped with a second determined heterologous viral envelope pseudotyping protein or viral envelope pseudotyping proteins distinct from said first heterologous viral envelope pseudotyping protein(s); wherein said first and second viral envelope pseudotyping protein(s) do not sero-neutralize with each other and are suitable for in vivo transduction of mammalian cells, especially of human cells.

[0138] The expression "combination of compounds" or alternatively "kit of compounds" means that the lentiviral vector particles constituting active ingredients of the kits or combinations, are provided as separate compounds in said kit or combination, and are intended for separate administration to a host, especially separate administration in time. Accordingly the invention enables to perform a prime-boost administration in a host in need thereof, where the first administration step elicits an immune, especially cellular, immune response and the later administration step(s) boost(s) the immune reaction including the cellular immune response. For each step of administration, it is preferred that the pseudotyping envelope protein(s) of the vector particles is different than the one used in the other step(s). Accordingly, the separate compounds of the kit or combination of the invention have distinct particles at least due to the difference in their pseudotyping envelope proteins.

[0139] The compounds of the kit thus are provided separately in time to the host in need thereof, especially to a mammalian host, in particular a human host.

[0140] Accordingly, said lentiviral vectors can be provided in separate packages or can be presented in a common package for a separate use thereof.

[0141] Therefore, the notice included in the packages and comprising the directions for use, may indicate that said lentiviral vector particles which are pseudotyped with distinct pseudotyping envelope protein or pseudotyping envelope proteins are for separate administration in time, especially for priming and subsequently boosting an immune reaction in a host.

[0142] In accordance with the invention, in the combination of compounds it is provided lentiviral vector particles which are pseudotyped with a first determined heterologous viral pseudotyping envelope protein, or viral pseudotyping envelope proteins, and lentiviral viral vector particles which are pseudotyped with a second determined heterologous viral pseudotyping envelope protein or viral pseudotyping envelope proteins. Accordingly, said first and second heterologous viral envelope protein(s) are different and in particular are originating from different virus strains. Thus, the lentiviral vector particles contained in the separate compounds of the of the kit of compounds of the invention are distinct from each other, at least due to the particular pseudotyping envelope protein(s) used for pseudotyping the vector particles.

[0143] In a particular embodiment of the invention, the combination of compounds comprises a third or a further type of lentiviral vector particles wherein the pseudotyping envelope protein(s) of the third lentiviral vector is different from said first and second pseudotyping envelope protein(s) and especially originates from a different virus strain.

[0144] When particles are successively administered which have different pseudotyping envelopes, the following order of administration with respect to said envelopes could be preferred: Indiana; New Jersey; Isfahan; SVCV/Cocal. Because Cocal pseudotyped lentiviral vectors seroneutralize several other envelopes, it is preferable, in the vaccination chronology, when Cocal envelopes are to be used in the preparation of particles, to administer particles pseudotyped with them as the last one in the administration regimen.

[0145] Apart from their pseudotyping envelope protein(s), the lentiviral vectors of the invention may be identical and especially may have identical vector genomes.

[0146] Alternatively, their vector genomes may be different, provided they carry the same recombinant determined polynucleotide (also designated as transgene), especially the same recombinant polynucleotide.

[0147] In another embodiment of the invention, the vector genomes of the lentiviral vectors are different by having at least one different recombinant polynucleotide, provided at least one of said different polynucleotides encodes polypeptide(s) having common antigenic determinant(s), or common epitope(s). Hence the different polynucleotides may be variants from each other that encode identical or variant polypeptides or may include sequences encoding different polypeptides.

[0148] A particular kit of compounds comprises lentiviral vectors wherein in at least one of the separate compounds, the vectors are pseudotyped with recombinant pseudotyping envelope protein(s) comprising combined domains or fragments originating from different envelope protein(s) of different viruses, especially of different genus of different species of VSV.

[0149] In a particular embodiment of the invention, at least one the first, second and if any third or further pseudotyping envelope protein(s) is (are) recombinant envelope protein(s) comprising the export determinant of the VSV-G of Indiana strain.

[0150] The export determinant of the VSV-G of the Indiana strain is a polypeptide encoded by the cytoplasmic fragment of the open reading frame of the envelope.

[0151] The export determinant of the VSV-G of the Indiana strain is a polypeptide comprising or having amino acid sequence YTDIE in the cytoplasmic tail (Nishimua N. et al. 2002).

[0152] Said recombinant envelope protein(s) may comprise the cytoplasmic tail of the VSV-G of an Indiana strain which is the intracellular portion of VSV-G delimited by a hydrophobic transmembrane domain.

[0153] A particular kit of compounds comprises lentiviral vectors wherein one or two or more of them is (are) pseudotyped with recombinant envelope protein(s) comprising the cytoplasmic domain of the indiana VSV and the ectodomain of a strain of a different VSV serotype. The transmembrane domain may also be the one of the Indiana VSV-G.

[0154] A particular kit of compounds comprises lentiviral vectors wherein one or both of them is (are) pseudotyped with recombinant envelope protein(s) comprising the transmembrane domain and the cytoplasmic domain of the indiana VSV and the ectodomain of the New-Jersey VSV.

[0155] Appropriate other modifications encompass mutations, especially point mutations, that improve pseudotyping. Such mutations for the VSV-G proteins may be carried out in the transmembrane domain by substituting or deleting one or several amino acid residues. Other examples of appropriate mutations are disclosed in Fredericksen B. L. et al (1995) or Nishimura N. et al (2003).

[0156] It is also especially possible to modify the glycosylation status of the VSV-G, in order to improve transduction efficiency of the lentiviral vector pseudotyped with these VSV-G proteins, when administered to a host.

[0157] VSV-G proteins from various strains of VSV are disclosed in the figures and their sequences can also be derived from databases, especially from GenBank. Especially the VSV-G proteins of Indiana and New-Jersey strains may be obtained by reference to the sequences disclosed as GenBank #AF170624 for New-Jersey VSV-G or GenBank #M11048 for Indiana strain.

[0158] Considering the glycoproteins of the New-Jersey and Indiana strains of VSV, it has been proposed that glycosylation at two asparagine residues (N180 and N336) favour the efficient pseudotyping of lentiviral vectors. This particular feature may be applied in the preparation of the lentiviral vectors of the invention.

[0159] The following constructs encoding VSV-G derived envelope proteins are particular examples of constructs for use in the preparation of the combination of lentiviral vector particles of the invention and are described in WO 2009/019612.

[0160] A VSV-G Indiana gene which is codon optimized as shown in SEQ ID No 76. A particular encapsidation plasmid is pThV-VSV.G (IND-CO) deposited at the CNCM (Paris, France) on Oct. 10, 2007, under number 1-3842 or in an alternative version of the plasmid construct, on Jul. 31, 2008, under number CNCM 1-4056 is suitable for use in preparing pseudotyped particles with an envelope from VSV-G Indiana New-Jersey. Other constructs may be derived from this particular plasmid, especially by substituting the promoter for a promoter among those listed in the present application.

[0161] A VSV-G New-Jersey gene codon optimized is disclosed in SEQ ID No 78. A particular encapsidation plasmid is pThV-VSV.G (NJ-CO) deposited at the CNCM (Paris, France) on Oct. 10, 2007, under number 1-3843 or in an alternative version of the plasmid construct, on Jul. 31, 2008, under number CNCM I-4058 is suitable for use in preparing pseudotyped particles with an envelope from VSV-G Indiana New-Jersey. Other constructs may be derived from this particular plasmid, especially by substituting the promoter for a promoter among those listed in the present application.

[0162] Other envelope genes suitable to carry out the invention having codon optimized sequences are illustrated in WO 2009/019612 and especially encompass VSV-G Chandipura gene and its expression product, VSV-G Cocal gene and its expression product, VSV-G Piry gene and its expression product, VSV-G Isfahan gene and its expression product, VSV-G Spring viremia carp virus gene and its expression product. A particular encapsidation plasmid, containing an envelope gene for VSV-G Cocal, is pThV-VSV.G (COCAL-CO) deposited at the CNCM (Paris, France) on Jul. 31, 2008, under number CNCM 1-4055. Another particular encapsidation plasmid, containing an envelope gene for VSV-G Isfahan, is pThV-VSV.G (ISFA-CO) deposited at the CNCM (Paris, France) on Jul. 31, 2008, under number CNCM 1-4057. Another particular encapsidation plasmid, containing an envelope gene for VSV-G Spring viremia carp virus, is pThV-VSV.G (SVCV-CO) deposited at the CNCM (Paris, France) on Jul. 31, 2008, under number CNCM 1-4059. These constructs are disclosed in patent application WO2009/019612.

[0163] Fusion envelope proteins, especially fusion proteins involving several different fragments of VSV-G proteins of different viruses and to the nucleic acid constructs encoding such proteins are used as alternative embodiments and are also disclosed in WO 2009/019612. A particular fusion envelope is the fusion between the ectodomain of the New-Jersey envelope protein and the transmembrane domain and cytoplasmic domain of the Indiana envelope protein as illustrated in the herein provided sequences.

[0164] Another fusion envelope protein suitable to perform the invention comprises the ectodomain of one VSV-G protein selected among VSV-G Chandipura, VSV-G Cocal, VSV-G Pyri, VSV-G Isfahan, or VSV-G SVCV and the tranmembrane and cytoplasmic domains of VSV-G Indiana. A nucleic acid molecule encoding said fusion protein is advantageously a codon optimized nucleic acid. Nucleic acid encoding the fusion protein are also described as SEQ ID No 77, 79, 81, 83 85, 87, 89.

[0165] In a particular embodiment of the invention, a combination of compounds is provided, wherein the lentiviral particles of the separate compounds encode (i) a polypeptide of the CSP antigen or (ii) a polypeptide of the CSP antigen devoid of the GPI-anchoring motif (CSP deltaGPI) or a CSP protein truncated in the N-terminal end (CSP NTer or also CSP delta SP).

[0166] In a particular embodiment, these compounds or some of them further encode at least one additional polypeptide of an antigen of the malaria parasite chosen in the groups disclosed herein, the distinct polypeptides of said antigens being either expressed from the same lentiviral particles or from distinct lentiviral particles.

[0167] In another particular embodiment of the invention, these compounds or some of them further encode at least one additional polypeptide of an antigen of the malaria parasite chosen in the groups disclosed herein.

[0168] The invention concerns especially lentiviral vector particles or a combination of compounds as herein defined for the prophylactic immunization against malaria parasite infection or against parasite-induced pathology in a mammalian host, especially in a human host.

[0169] Accordingly, the lentiviral vector particles, compositions comprising the same or the combination of compounds of the invention, when administered to a host in needs thereof, especially to a mammalian in particular to a human host, elicit an immune response, encompassing activation of naive lymphocytes and generation of effector T-cell response and generation of immune memory antigen-specific T-cell response against antigen(s) of the malaria parasite. The immune response may either prevent the infection by the malaria parasite when such parasite is inoculated as sporozoite to the host or may prevent the onset or the development of a pathological state resulting from inoculation of malaria parasite in the form of sporozoite or prevent the onset or the development of the consequences of the generation of further forms of said parasite such a merozoite forms.

[0170] Accordingly, the lentiviral vector particles or the combination of compounds of the invention are suitable for prevention, control or inhibition of the onset of the pathology caused by inoculation of the parasite or by the induction of the exo-erythrocytic i.e., hepatic, stage of the cycle of the malaria parasite and in an advantageous embodiment are suitable to prevent, alleviate or inhibit the onset or development of the erythrocytic cycle of said parasite. Advantageously, it has been observed that the lentiviral vector particles of the invention used in a prime-boost regimen of administration enable the development of a protective immunity and especially enable a sterilizing protection against the malaria parasite-induced pathology. Such a sterilizing protection may result from controlling the consequences of the infection at the stage of liver infection, if not before, in cycle of the parasite.

[0171] In a particular embodiment of the invention, a composition of lentiviral vector particles is prepared wherein said lentiviral vector particles are formulated with a suitable administration vehicle for use for prophylactic immunization against malaria parasite infection or against parasite-induced pathology in a mammalian host, especially in a human host.

[0172] Physiologically acceptable vehicles may be chosen with respect to the administration route of the immunization composition. In a preferred embodiment administration may be carried out intramuscularly or, for children intranasally.

[0173] Accordingly, a combination of compounds can comprise separately provided compositions of lentiviral vector particles wherein each separate composition of the combination or kit of compounds comprises lentiviral vector particles, pseudotyped with a determined heterologous viral pseudotyping envelope protein or proteins, and wherein said pseudotyping envelope proteins do not cross-react with to sero-neutralize the pseudotyping envelope proteins of the lentiviral vector particles of another composition of the combination or kit of compounds.

[0174] Accordingly, such compositions or combination of compounds of said compositions are used for prophylactic immunisation against malaria parasite infection or against parasite-induced pathology in a mammalian host, especially in a human host, said use involving an immunisation pattern comprising administering an effective amount of the lentiviral particles to prime the cellular immune response of the host and later in time administering an effective amount of lentiviral particles to boost the cellular immune response of the host, and optionally repeating (once or several times) said administration step for boosting, wherein the lentiviral particles administered in each of the priming or boosting steps are pseudotyped with distinct pseudotyping envelope protein(s) which do not cross-neutralise with each other, and wherein said priming and boosting steps are separated in time by at least 6 weeks, in particular by at least 8 weeks.

[0175] In the examples which follow where mice models have been treated according to the prime-boost regimen with lentiviral vector particles of the invention, it has been shown by the inventors that mice immunized according to such a regimen and challenged 6 months after the last immunization step still exhibit a sterile protection for a significant proportion of the vaccinated mice (more than 40%) which illustrates that the lentiviral vector particles of the invention elicit a long-lasting sterile protection in a host, and would therefore constitute a suitable compound for immunization especially in a human host.

[0176] The invention relates, in a particular embodiment, to the lentiviral vector particles or combination of compounds as defined herein, for the prophylactic immunization against malaria parasite infection or against parasite-induced pathology in a mammalian host, especially in a human host, in a dosage regimen comprising separately provided doses of said lentiviral particles wherein the dose intended for priming and boosting the cellular immune response is a moderate dose and the dose intended for boosting the cellular immune response is higher than the dose for priming.

[0177] Accordingly, the dose intended for priming and boosting the cellular immune response which is used in the administration pattern, comprises from 10⁷ TU to 10⁹ TU of viral particles when integrative vectors are used, the dose intended for children being in the range of 10⁷TU and for adults in the range of 10⁹TU. The dose intended for priming and boosting comprises from 10⁸ to 10¹⁰ of lentiviral particles when integrative-incompetent vectors are used.

[0178] The lentiviral vector particles or the combination of compounds of the invention is especially used in a particular embodiment for the prophylactic immunization against malaria parasite infection or against parasite-induced pathology in mammalian, host, especially in a human host, in a dosage and administration regimen which is suitable to obtain at least one of the following effects in the host:

[0179] eliciting sterile protection against malaria parasite infection, especially by Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium knowlesi or Plasmodium ovale in a human host;

[0180] inhibiting extracellular forms of malaria parasite;

[0181] preventing hepatocytes infection by malaria parasite or inhibition of liver stage amplification of infection;

[0182] eliciting a specific T-cell immune response against malaria parasite antigen(s), especially a CD8+ T-cell response and/or a specific CD4+ T-cell response;

[0183] eliciting a B-cell response against parasite antigen(s);

[0184] controlling parasitemia so as to reduce or alleviate the effects of infection by the malaria parasite;

[0185] eliciting a protective cellular immunity against the infection by the parasite or against the parasite-induced pathology;

[0186] eliciting memory T-cell immune response

[0187] eliciting earlier and higher rebound of the CD4+ and CD8+ T-cell response during infection by the malaria parasite;

[0188] eliciting earlier and strong CT (CD8+T) response by stimulating intra hepatic memory lymphocytes upon Plasmodium infection.

[0189] preventing malaria parasite escape from immune response thereby allowing long-term control of the infection by the malaria parasite.

[0190] Among the above targeted effects, a cellular immune response (T-cell immune response), particularly a CD8-mediated cellular immune response or a CD4-mediated cellular immune response i.e., an immune response which is mediated by activated cells harbouring CD8 or CD4 receptors, preferably Cytotoxic T lymphocytes (CTL) and memory T cell response are advantageously targeted when defining the immunization regimen of the lentiviral particles of the invention.

[0191] The immune response can also involve a humoral response i.e., antibodies, elicited by said lentiviral vector particles, produced against said at least one polypeptide of the lentiviral vector. In a particular embodiment, said humoral response is a protective humoral response. The protective humoral response results mainly in maturated antibodies, having a high affinity for their antigen, such as IgG. In a particular aspect, the protective humoral response is T-cell dependent. In a particular embodiment, the protective humoral response induces the production of neutralizing antibodies.

[0192] In a particular embodiment of the invention, the lentiviral vector of the invention, even when used in a form which has defective integrase, is able to elicit an early immune response. The expression "early immune response" refers to a protective immune response (protection against the parasite or against the parasite-induced pathology) that is conferred within about one week after the administration of the composition.

[0193] In another particularly advantageous embodiment, the immune response conferred by the lentiviral particles of the invention is a long-lasting immune response i.e., said immune response encompasses memory cells response and in particular central memory cells response; in a particular embodiment it can be still detected at least several months, (as illustrated for mice in the examples a protection is still obtained after at least 6 months after the administration of the particles) which allows to consider that the protection may last in a human host over several years following the administration.

[0194] When the immune response includes a humoral response, the long-lasting response can be shown by the detection of specific antibodies, by any suitable methods such as ELISA, immunofluorescence (IFA), focus reduction neutralization tests (FRNT), immunoprecipitation, or Western blotting.

[0195] In a particular embodiment, said immune response, either humoral or cellular, early immune response and/or long-lasting immune response, is elicited with the non-integrative gene transfer vector, after a single administration of the composition of the invention.

[0196] The invention also concerns the use of the lentiviral vector particles or the use of a combination of compounds according to the definition given herein, for the manufacture of an immunogenic composition for prophylactic immunisation against malaria parasite infection or against parasite-induced pathology in a mammalian host, especially in a human host.

[0197] The invention also concerns a method of providing immunization in a mammalian host, especially in a human host, comprising the step of administering the lentiviral vectors of the invention to elicit the immune response, and optionally repeating the administration steps one or several times, to boost said response, in accordance with the present disclosure.

[0198] In a particular embodiment of the invention, the lentiviral vector particles or the combination of compounds may be used in association with an adjuvant compound suitable for administration to a mammalian, especially a human host, and/or with an immunostimulant compound, together with an appropriate delivery vehicle.

[0199] The compositions quoted above can be injected in a host via different routes: subcutaneous (s.c.), intradermal (i.d.), intramuscular (i.m.) or intravenous (i.v.) injection, oral administration and mucosal administration, especially intranasal administration or inhalation. The quantity to be administered (dosage) depends on the subject to be treated, including considering the condition of the patient, the state of the individual's immune system, the route of administration and the size of the host. Suitable dosages range expressed with respect to the content in equivalent p24 antigen of vector particles (for HIV-1 lentiviral vectors) and can be determined.

[0200] Other examples and features of the invention will be apparent when reading the examples and the figures which illustrate the preparation and application of the lentiviral vector particles with features that may be individually combined with the definitions given in the present description.

LEGEND OF THE FIGURES

[0201] FIG. 1. Nonintegrative lentiviral vector-based vaccination confers total inhibition of liver stage development. A. Study design. Naive mice were primed at week 0 with 100 ng of TRIP.NI CS particles pseudotyped with the VSV-G Indiana (VSV-G Ind) envelope and then boosted at week 8 with 1500 ng of TRIP.NI CS particles pseudotyped with the VSV-G New Jersey (VSV-G NJ) envelope. One group of vaccinated mice were challenged with 80.000 sporozoites (spz) of Plasmodium yoelii (17XNL-gfp.sup.+ strain) and protective efficacy was measured by quantifying the liver parasite load 40 hours later. A second group of vaccinated mice were challenged with 500 spz of Plasmodium yoelii (17XNL-gfp.sup.+ strain) and protective efficacy was evaluated by monitoring blood stage parasitemia every other day from day 3 post injection until day 14 by Giemsa-stained blood smears. In the two cases, the challenges were performed one month after the last immunization. B. Results of the parasite loads quantified using real-time RT-PCR for P. yoelii 18S rRNA in the livers of challenged mice. Data are presented as the number of copies of plasmodium 18S rRNA detected in individual control mice (n=5) and vaccinated mice (n=4). Mean+/-SD of duplicate is shown. C. Results of the monitoring of blood stage parasitemia. 0 indicates absence of parasites, + indicates presence of parasites.

[0202] FIG. 2. A. Study design. Mice primed with 100 ng of TRIP.NI CS particles pseudotyped with the VSV-G Indiana (VSV-G Ind) envelope and boosted 8 weeks later with 1500 ng of TRIP.NI CS particles pseudotyped with the VSV-G New Jersey (VSV-G NJ) envelope received a third immunization dose 5 months later with 1500 ng of TRIP.NI CS particles pseudotyped with the VSV-G Cocal. Vaccinated mice were challenged one month later with 500 spz of Plasmodium yoelii (17XNL strain) and protective efficacy was evaluated by monitoring blood stage parasitemia every other day from day 3 post injection until day 16 by Giemsa-stained blood smears. B. Percentage of mice completely protected against sporozoite challenge after a nonintegrative lentiviral vector-based regimen. C. Means of parasitemia of naive mice (CO-black curve), vaccinated mice completely protected (VAC-light grey curve) and vaccinated mice partially protected (VAC-grey curve) are depicted. D. Means of parasitemia from naive mice (CO-- black), vaccinated mice partially protected (VAC-grey) and vaccinated mice completely protected (VAC-grey), 10 days after the challenge.

[0203] FIG. 3. Gross morphology of spleens and livers from mice vaccinated (VAC) or not (CO) at the final killing (3 weeks post-challenge) with 500 sporozoites of Plasmodium yoelii.

[0204] FIG. 4. CS protein-specific T cell responses from splenocytes of vaccinated mice 3 weeks after challenge. Ex vivo IFNquadrature ELISPOT was carried out using splenocytes from vaccinated mice harvested 3 weeks after the challenge with Plasmodium yoelii. Splenocytes were stimulated with synthetic peptides representing CD8.sup.+ or CD4.sup.+ defined epitopes. Data are expressed as mean+/-SD of spot forming cells (sfc) of duplicate wells. n=5 in the protected group and n=3 in the unprotected group. *: different from unprotected group p<0.05.

[0205] FIG. 5. Optimized non integrative lentiviral vectors confer long term sterile protection against malaria (a) Vaccine schedule. Mice were primed with 100 ng of TRIP.NI CSP particles pseudotyped with the VSV-G Indiana envelope and boosted 8 weeks later with 1500 ng of TRIP.NI CSP particles pseudotyped with the VSV-G New Jersey envelope. Five months later, they received a third injection of TRIP.NI CS particles (1500 ng) pseudotyped with the VSV-G Cocal envelope. Animals were challenged six months later with 500 sporozoites of Plasmodium yoelii (17XNL strain) and protective efficacy was evaluated by monitoring blood stage parasitemia every other day from day 3 post injection until day 16. (b) Means of parasitemia from naive mice (CO-- black), vaccinated mice partially protected (VAC-light grey (middle)) and vaccinated mice completely protected (VAC-grey (right)), 10 days after the challenge. (c) Tetramer analysis of the % of CSP-specific CD8.sup.+ T cells from the spleen, the bone marrow and the liver of mice at the final killing (3 weeks post-challenge).Black bars indicate vaccinated mice partially protected and white bars indicate vaccinated mice completely protected. (d) INF-g ELISPOT quantification of CSP-specific CD8+ T cells in the spleen, the bone marrow and the liver of mice. * P<0.05 (Student's t-test)

[0206] FIG. 6. Hep17-specific T cell responses induced by nonintegrative lentiviral vectors. Naive mice (n=5/group) were immunized or not (-) i.p. with a single injection of various doses (100 or 600 ng) of nonintegrative lentiviral vectors coding for Hep17. At 11 days post-immunization, Hep17-specific cellular immune responses against the CD8+ T cell epitopes (A) and the CD4.sup.+ T cell epitopes (B) were assessed by IFN-γ ELISPOT. SFC, spot-forming cells.

[0207] FIG. 7. Hep17-specific T cell responses induced by integrative lentiviral vectors. Naive mice (n=5/group) were immunized (or not: -) i.m. with a single injection of integrative lentiviral vectors (1×10⁷ TU) coding for Hep17. At 11 days post-immunization, Hep17-specific cellular immune responses against the CD8.sup.+ T cell epitopes (A) and the CD4.sup.+ T cell epitopes (B) were assessed by IFN-γ ELISPOT. SFC, spot-forming cells.

[0208] FIG. 8. CS-- and Hep17 specific T cell responses elicited after coimmunizations with lentiviral particles. Naive mice (n=5/group) were immunized i.m. with a single injection of integrative lentiviral vectors (1×10⁷ TU) coding for CS (named CSP in figure A and B) or Hep17 (named Hep17 in figure C and D). For coimmunization experiments, naive mice were injected into one quadriceps with TRIP.I CS and into the opposite quadriceps with TRIP.I Hep17 particles (named CSP+Hep in figure A,B,C,D). At 11 days post-immunization, CS-specific cellular immune responses (A) and Hep17-specific cellular immune responses (C) were assessed by IFN-γ ELISPOT. SFC, spot-forming cells. For in vivo cytotoxic assays, immunized mice were injected at day 11 with target cells pulsed with CS peptides (C) or Hep17 peptides (D). Percentages of specific killing were determined 18 hours later, as described in Material and Methods section.

[0209] FIG. 9. A single dose of nonintegrative lentiviral vector coding for MSP1₄₂ elicits a strong and specific antibody response. A. Groups of adult mice (n=5) were immunized intraperitoneally with graded doses of TRIP.I MSP1₄₂. After 21 days, pooled sera (5 mice per group) were assessed for the presence of MSP-1₁₉-specific antibodies. B. Mice were primed with 100 ng of TRIP.I MSP1₄₂ particles pseudotyped with the VSV-G Indiana envelope. 3 months later, mice were boosted with 1000 ng of TRIP.NI MSP1₄₂ particles pseudotyped with the VSV-G cocal envelope. Results are the mean titers of MSP-1₁₉-specific antibodies detected in the sera of mice 3 weeks after the last immunization.

[0210] FIG. 10. Alignments of Plasmodium CSP proteins and sequence of a consensus.

[0211] FIG. 11. Restriction map of plasmid pTRIP-DeltaU3-CMV-eGFP. SEQ ID No 33

[0212] FIG. 12. Restriction map of plasmid pTRIP-ΔU3-CMV-MSP1₄₂ CO--WPRE (CNCM 1-4303 or SEQ ID No 34).

[0213] FIG. 13. Restriction map of plasmid pTRIP-ΔU3-CMV-Hep17 CO--WPRE (CNCM 1-4304 or SEQ ID No 37).

[0214] FIG. 14. Restriction map of plasmid pTRIP-ΔU3-CMV-Hep17 ASP CO--WPRE (CNCM 1-4305 or SEQ ID No 40).

[0215] FIG. 15. Restriction map of plasmid pTRIP-ΔU3-CMV-CSP CO--WPRE (CNCM 1-4306 or SEQ ID No 43).

[0216] FIG. 16. Restriction map of plasmid pTRIP-ΔU3-CMV-CSP ASP CO--WPRE (CNCM 1-4307 or SEQ ID No 45).

[0217] FIG. 17. Restriction map of plasmid pTRIP-ΔU3-CMV-CSP ΔGPI CO--WPRE (CNCM 1-4308 or SEQ ID No 47).

EXAMPLES

[0218] With a view to assess whether lentiviral vectors may represent an alternative strategy, a nonintegrative lentiviral vector coding for a truncated form of the circumsporozoite (CS) protein of Plasmodium yoelii (TRIP.NI CS) was designed and assayed in an animal mice model relevant for malaria. The CS protein is distributed uniformely over the surface of sporozoites and is also detected in infected liver cells⁴, 5. Thus, the induction of humoral immune responses against the CS protein reduces the hepatocyte infectivity, whereas the cellular immune responses against this antigen kill parasite-infected hepatocytes. This concept was recently supported by an elegant study that demonstrated that the CS protein was the main target of protective immunity in the irradiated sporozoite immunization model⁶. Moreover, among all the vaccine candidates tested so far in clinical trials, only the CS protein-based vaccine RTS, S has been shown to reduce significantly malaria incidence and cases of severe malaria in endemic countries⁷, 8.

[0219] In order to elicit optimal immune responses against the CS protein, we combined three strategies: 1) to increase the level of antigen expression in transduced cells, we inserted in the vector backone a mamalian codon-optimized sequence of the CS protein under the control of the strong cytomegalovirus promoter and we added downstream the transgene the woodchuck post-transcriptional regulatory element sequence to increase mRNA stabilization and export to the cytoplasm; 2) we deleted the GPI anchoring sequence located at the 3' end of the cs gene since deletion of GPI-anchoring motif has been shown to improve the immunogenicity of the CS protein⁹ 3) to increase the specific immune response, and in particular, to protect mice from infection by sporozoite challenge, mice received LV-based boosters. To circumvent the presence of neutralizing anti-envelope antibodies induced after the first immunization, lentiviral particles used for boost immunizations were pseudotyped with VSV-G envelopes from non-cross-reactive serotypes (VSV-G Indiana for the prime, VSV-G New Jersey and Cocal for the first and the second boost, respectively).

[0220] In a first series of experiment, mice were primed with a moderate dose of TRIP.NI CS and boosted 8 weeks later with a high dose of TRIP.NI CS (FIG. 1a). To evaluate protection induced by this prime-boost regimen, BALB/c mice were challenged with 80.10³ sporozoites of Plasmodium yoelii (17XNL gfp+ strain), the invasive form of the parasite present in the mosquitoe. The challenge was performed 4 weeks after the completion of immunization regimen. Forty hours after the challenge, the level of inhibition of liver stage development was determined by quantifying plasmodial 18S rRNA in the livers of mice. For this purpose, liver-extracted RNA was used for real-time PCR amplification of the plasmodial 18S rRNA sequences, using the EXPRESS One-Step SYBR® GreenER® kit (invitrogen) and specific primers for the amplification of the 18S rRNA of Plasmodium yoelii. As shown in FIG. 1b, the inhibition of liver stage development of the parasite was complete for all immunized mice, i.e., no parasite 18S rRNA could be detected by quantitative RT-PCR. In parallel experiments, protection was also assessed by examining blood smears of immunized mice, which were challenged with 500 Plasmodium yoelii sporozoites, for the occurrence of erythrocytic stages. Peripheral blood smears were obtained daily from day 3 to 14 post-challenge, stained with Giemsa and examined by microscopy, to determine whether immunized mice became parasitemic, i.e., failed to develop protection. As shown in FIG. 1c, complete protection occurred in 60% of immunized mice.

[0221] In a second series of experiments, we added a third injection of TRIP.NI CS pseudotyped with a VSV-G Cocal envelope which does not cross-react whith antbodies directed against the Indiana and New Jersey serotypes. One month after the last boost, immunized mice were challenged intravenously with 500 sporozoites,(FIG. 2a). Protective efficacy was evaluated by monitoring blood stage parasitemia every other day from day 3 post injection until day 21 by Giemsa-stained blood smears. After 5 days, all naive mice exhibited patent blood stage parasitemia. By contrast, 62,5% of immunized mice showed sterile immunity (as defined by the absence of parasitemia over the following 21 days) (FIG. 2b). Moreover, as compared with naive mice, immunized mice that developped parasitemia displayed a significant delay in the course of erythrocyte invasion (FIG. 2c). At day 10 post-challenge, immunized mice partially protected showed a twofold reduction in the level of parasitemia compared to naive mice, demonstrating that in this case, the vaccine afforded also an immune control, albeit partial, of the parasite (FIG. 2d).

[0222] Hepatosplenomegaly is a prominent feature of malaria. We then performed a qualitative analysis of the organs from mice sacrified 3 weeks post-challenge. Naive mice infected with the parasite displayed dramatic splenomegaly (FIG. 3). Moreover, spleens and livers showed dark pigmentation resulting from the accumulation of hemozoin produced by the parasite during digestion of red blood cell hemoglobin. By contrast, the capacity of 5 out of 8 vaccinated mice to mount a sterile immune response coincided with preservation of livers and spleens that displayed normal size and pigmentation.

[0223] In an attempt to understand why 1/3 of immunized mice did not shown sterile protection, we evaluated the CS protein-specific immune responses in vaccinated animals sacrified at 3 weeks post-challenge. Challenged naive mice displayed no detectable CS protein-specific IFN-β producing T cells (data not shown). By contrast, in the vaccinated group, mice fully protected exhibited five to eight fold greater CSP-specific T cell responses as compared with mice vaccinated but partially protected, emphasizing the critical importance of the strength of T cell responses for immune control (FIG. 4).

[0224] Importantly, we also performed challenge experiments at 6 months after the last immunization. In this case, more than 40% of the vaccinated mice still failed to develop detectable parasitemia following challenge, illustrating the long-lasting sterile protection conferred by our vaccine strategy (FIG. 5).

[0225] Taken together, these data demonstrated that a prime-boost regimen based on non integrative lentiviral vectors can confer a high degree of protection against challenging infectious agents such as plasmodium.

[0226] Based on these results, we are currently developing a multi-stage vaccine approach. The rationale of this strategy is to improve the protective efficiency conferred by our vaccine approach by inducing a multi-immune response directed against antigens expressed in the liver stage and targeted by T-cell responses, as well as antigens expressed in the blood-stage and targeted by antibody responses. To this end, we have selected two pre-erythrocytic stage antigens (CS protein and Hepatocyte Erythrocyte Protein 17 kDa--HEP17) and one erythrocytic stage antigen (The 42-kDa fragment of the Merozoite Surface Protein 1--MSP-1₄₂). These antigens were selected because it has been shown that cytotoxic T cell responses specific of Hep17 are partially protective against sporozoite challenge and antibody responses specific of MSP-1₄₂ can also protect mice against a lethal challenge with blood-stage parasites¹⁰, 11. Lentiviral vectors coding for Hep17 or MSP-1₄₂, were constructed as detailed in the Material and Methods part. To evaluate the immunogenicity of a single injection of lentiviral particles expressing Hep17, groups of mice (n=5/group) were immunized with 100 ng (3,2×10⁷ TU) or 600 ng (1,9×10⁸ TU) of TRIP.NI Hep17 and specific immune responses were assayed by Elispot. As shown in FIG. 6, relatively weak CD8 and CD4 responses could be detected in the spleens of immunized mice after stimulation with the 9-mer and 15-mer previously described¹².

[0227] We also tested the immunogenicity of TRIP.I Hep17 lentiviral particles. Groups of mice (n=5) were immunized im with 1×10⁷ TRIP.I Hep17 particles. Hep-17-specific IFNg Elispot responses were evaluated 11 days later on splenocytes from immunized mice. As shown in FIG. 7, the most robust responses were detected against the CD4+ T cell epitopes (KL14 and EK15) and against one CD8+ T cell epitope (LA9). We also evaluated the T-cell responses obtained after co-immunization of TRIP.I Hep17 particles with TRIP.I CS particles. Mice received two injections: one injection of 1×10⁷ TRIP.I Hep17 particles in the left quadriceps and one injection of 1×10⁷ TRIP.I CS particles in the right quadriceps. In parallel, groups of mice were immunized with TRIP.I CS particles alone (1×10⁷ TU im) or TRIP.I Hep17 particles alone (1×10⁷ TU im). At day 11, one part of immunized mice were sacrified for Elispot experiments. There was no huge difference between the frequency of CS-specific IFNg T cells in mice immunized with TRIP.CS particles alone or with TRIP.I CS and TRIP.I Hep17 particles (FIG. 8A). To evaluate cytotoxic T cell response in immunized mice, we performed an in vivo cytotoxic assay (as described in Material and Methods). At day 11, groups of mice immunized with TRIP.I CS particles alone or coimmunized with TRIP.I CS and Hep17 particles were challenged by iv injection with target cells pulsed with CS peptides. As expected, mice immunized with TRIP.I CS particles lyzed efficiently target cells and we did not detect significant difference between group of mice immunized with TRIP.I CS particles alone and group of mice that received both TRIP.I CS and TRIP.I Hep17 particles (FIG. 8B). Taken together, these results demonstrated that TRIP.I Hep17 particles co-administered with TRIP.I CS particles did not significantly interfere with the CS-specific T cell response elicited by TRIP.I CS particles. We also evaluated the frequency of Hep17-specific IFNg T cells in mice immunized with TRIP.I Hep17 alone or co-administrated with TRIP.I CS particles. Frequencies of specific T cells responding to stimulation to the five 9-mer peptides (CD8+ T cell epitopes) were the same in the two groups, as well as those measured after stimulation with the KL14 epitope (CD4+ T cell epitope). Strikingly, the responses detected against the CD4+ T cell epitope EK15 were twice higher in mice co-immunized than in mice immunized with TRIP.I Hep17 alone (FIG. 8C). As shown in FIG. 8D, the cytotoxic capacity of T cells against Hep17 peptides-pulsed targets were also greatly increased in mice co-immunized with TRIP.I Hep17 and TRIP.I CS particles. Collectively, these data demonstrate that CS-specific immune response enhances cytotoxic T cell responses specific for Hep17.

[0228] We next evaluated the ability of lentiviral vectors to initiate a B cell response against the blood stage malaria antigen merozoite surface protein-1 (MSP1). Mice (n=5) were immunized with various doses of integrative lentiviral vectors coding for the 42-kDa region of MSP1 from Plasmodium yoelii (TRIP.I MSP1₄₂) fused to at the N terminus to the secretory signal of the calreticuline. Three weeks after immunization, pooled sera collected from each group of immunized mice were tested for the presence of total anti-MSP1 antibodies directed against the protective C-terminal 19-kDa region (MSP-1₁₉)¹³, 14 of the antigen. As shown in FIG. 9A, mice immunized with a dose as low as 1×10⁶ TU displayed detectable levels of anti-MSP-1₁₉ antibodies and immunizations with 1×10⁷ TU of this vector induced a strong secretion of anti-MSP-1₁₉ Ig with a mean titer reaching 2×10³. To know whether anti-MSP1 response conferred by lentiviral vector immunization could be enhanced by a second immunization, mice immunized with 100 ng of TRIP.I MSP1₄₂ particles pseudotyped with VSV-G Indiana envelope were boosted 3 months later with 1000 ng of TRIP.NI MSP1₄₂ particles pseudotyped with the VSV-G Cocal envelope (FIG. 9B). 3 weeks after the last immunization, the levels of anti-MSP-1₁₉ antibodies in prime-boosted mice reached a mean value of 4×10⁵ whereas the titer in the plasma of mice solely primed was 2×10⁴. In conclusion, immunization with integrative lentiviral vectors can induce potent anti-MSP-1₁₉ Ig that have been shown to be protective against infection of red blood cells by parasites.

MATERIAL AND METHODS

[0229] Animals And Parasites.

[0230] Balb/c Ola Hsd (six-week-old female) were purchased from Harlan Laboratories (Gannat, France). All animal experiments were conducted in accordance with the guidelines of Animal Care at the Pasteur Institute. Infection experiments were performed with the Plasmodium yoelii (17XNL strain) wild-type or genetically modified to express the green fluorescent protein, allowing the detection of oocysts and sporozoites in living mosquitoes. Plasmodium yoelii was maintained by alternate cyclic passages in Anopheles stephensi and Balb/c mice. Mosquitoes were reared at the Center for Production and Infection of Anopheles (CEPIA) of the Pasteur Institute using standard procedures.

[0231] Plasmid Vectors Construction.

[0232] The mammalian codon optimized form of the gene coding for the full-length of the Py CS protein (amino acids 1-367; GenBank Accession No. M58295) was synthesized by Geneart. Since deletion of the GPI-anchoring motif has been shown to improve the immunogenicity of the CS protein, we constructed a codon optimized form of the cs gene deleted of the sequence encoding the last 11 amino acids. This sequence was obtained by PCR amplification of a fragment of the codon optimized cs gene using the following oligonucleotides (Sigma-Proligo): (forward) 5' GGTACCGGATCCGCCACCATGAAGAAA TGCACC-3' (underlined is the BamHI site); (reverse) 5'-AGCTCGAGTCATCACAGGCTGTTGGACACGATGTTGAAGATGC-3' (underlined is the XhoI site). The resulting amplicon was cloned in a pCR 2.1-TOPO plasmid (Invitrogen) and sequenced (plasmid referred as pCR 2.1-TOPO CS). The pTRIP CS vector plasmid was generated by replacing the GFP sequence from pTRIP CMV-GFP-WPRE digested BamHI/XhoI by the truncated codon-optimized CS sequence obtained after a BamHI/xhoI digestion of the pCR 2.1-TOPO CS. For pTRIP Hep17, a mammalian codon-optimized sequence (Geneart) of the Py Hep17 gene (GenBank Accession No. U43539) including a kozak sequence and flanked of a BamH1 site in 5' and a XhoI site in 3' was cloned in pTRIP CMV-WPRE digested BamH1/XhoI. For MSP1 construct, a composite mammalian codon optimized sequence (Geneart) was designed to include: a sequence coding for the secretion signal of the calreticuline (MLLSVPLLLGLLGLAVA) fused to the codon optimized sequence of the Py MSP1₄₂ (GenBank Accession No. J04668). The entire sequence digested BamH1/XhoI was cloned in pTRIP CMV-WPRE digested BamH1/XhoI.

[0233] Sequences of the pTRIP vectors are respectively designated as: SEQ ID NO 34, 37, 40, 43, 45 and 47.

[0234] Lentiviral Vector Production.

[0235] Vector particles were produced by transient calcium phosphate co-transfection of 293T cells with the vector plasmid pTRIP CS, a VSV-G envelope expression plasmid (pHCMV-G) and the pD64V encapsidation GAG POL plasmid for the production of integration-deficient vectors (the D64V substitution in the catalytic domain of the integrase blocks the DNA cleaving and joining reactions of the integration step) as previously described¹⁵. Quantification of the p24 antigen content of concentrated vector particles was performed with a commercial HIV-1 p24 enzyme-linked immunoabsorbent assay (ELISA) kit (Perkin Elmer Life Sciences). Vector titers of TRIP.I and TRIP.NI particles were determined by transducing HeLa cells treated with aphidicolin (SIGMA) and performing a quantitative PCR as previously described¹⁵. The titers of integrative and nonintegrative lentiviral vectors were similar according to p24 content and quantitative PCR measured in growth-arrested cells.

[0236] Mice Immunization and Challenge.

[0237] Six-week-old BALB/c mice were intraperitoneally (i.p.) immunized with 100 ng of TRIP.NI CS vector particles pseudotyped with the VSV-G Indiana envelope, diluted in 0.1 ml Dulbecco's phosphate-buffered saline. Eight weeks later, mice were boosted i.p. with 1500 ng of TRIP.NI CS vector particles pseudotyped with the VSV-G New Jersey envelope. Challenge of the immunized and the control mice consisted of the injection of 80,000 sporozoites intravenously 4 or more weeks after the last immunization. The outcome of the challenge was determined by measuring the parasite burden in the liver of mice by using a quantitative real-time RT-PCR method, as detailed later. We also determined, in control and immunized groups of mice, whether or not mice developed parasitemia after i.v. inoculation of 500 sporozoites, by microscopic examination of Giemsa-stained thin blood smears obtained daily, from the third day after challenge up to day 14. Briefly, a small drop of blood from challenged mice was placed on a microscope slide. The drop was smeared by using a second slide, air-dried, and fixed in 100% methanol for 30 seconds. Fixed slides were stained for 30 minutes in a fresh solution of 10% Giemsa (Reactfs RAL) diluted in water (Volvic), rinsed with water and dried in air. The slides were observed with x100 oil immersion objective.

[0238] Quantification of P. yoelii by Real-Time RT-PCR.

[0239] Quantification of the parasite loads in the liver of the challenged mice was performed as previously described¹⁶ with some modifications. 40 hours after challenge, livers were harvested and RNA was extracted with the RNeasy mini kit (Qiagen). 2 μg of RNA was used for the quantification of parasite specific 18S rRNA. The reaction of real-time RT-PCR was carried out with the EXPRESS One-Step SYBR® GreenER® kit (invitrogen) and specific primers for the amplification of the 18S rRNA of P. yoelii. The sequences of the primers (Sigma-Proligo) are: 5'-GGGGATTGGTTTTGACGTTTTTGCG-3' (forward primer) and 5'-AAGCATTAAATAAAGCGAATACATCCTTAT-3' (reverse primer). Experiments were performed with a LightCycler® apparatus (Roche diagnostics). The quantity of parasite RNA copies was assessed by extrapolation of threshold fluorescence values onto an internal standard curve prepared from serial dilutions of a plasmid construct (pCR 2.1-TOPO plasmid-Invitrogen) containing the 18S cDNA PCR-amplified fragment of the parasite.

[0240] Elispot Assay.

[0241] Nitrocellulose microplates (MAHA S4510, Millipore) were coated with capture antibody (Mouse IFNg Elispot pair, BD Pharmingen) and blocked with complete medium composed of RPMI 1640 Glutamax supplemented with 10% FCS, antibiotic, Hepes, non-essential amino-acids, b-mercaptoethanol and sodium pyruvate. Splenocytes from vector-immunized mice were added to the plates in triplicates at 0,125×10⁶ cells/well. For quantification of CS-specific CD8.sup.+ T cell reponses, splenocytes were incubated with 2 μg/ml of the peptides (PolyPeptide Laboratories France) SYVPSAEQI (Py CS_280-288) or IYNRNIVNRL (Py CS_58-67). To evaluate the CS-specific CD4.sup.+ T cell responses, splenocytes were incubated with 2 μg/ml of the peptides SYVPSAEQILEFVKQI (Py CS_280-295). Twenty hours later, spots were revealed with the biotin-conjugated antibody (Mouse IFNg Elispot pair, BD Pharmingen) followed by streptavidin-AP (Roche) and BCIP/NB substrate solution (Promega). Spots were counted using a Bioreader 2000 (Biosys, Karben, Germany) and results were expressed as IFNγ spot-forming cells (sfc) per million splenocytes. The same protocol was applied for quantification of Hep17-specific T cell responses. Peptides used for stimulation in Elispot and in vivo cytotoxic assay are summarized in Table 1.

[0242] In vivo cytotoxic assay. For target cell preparation, splenocytes from naive mice were labelled with various concentrations (high, 5 μM; Low, 1 μM) of CFSE (carboxyfluorescein-diacetate succinimydyl ester, Vybrant CFDA-SE cell-tracer kit, Molecular Probes). Splenocytes labelled with high concentrations of CFSE were pulsed with combination of peptides at 5 μg/ml. The control population stained with low doses of CFSE was incubated in medium without peptides. Each mouse received 10⁷ CFSE-labelled cells of a mix containing an equal number of cells from each fraction, through the retroorbital vein. After 15-18 h, single-cell suspensions from spleen were analyzed by flow cytometry (Becton Dickinson, CellQuest software). The disappearance of peptide-pulsed cells was determined by comparing the ratio of pulsed (High CFSE fluorescence intensity) to unpulsed (Low CFSE fluorescence intensity) populations in immunized versus naive mice. The percentage of specific killing was established according to the following calculation: (1-((CFSE_low naive/CFSE_high naive)/(CFSE_low immunized/CFSE_high immunized)))*100.

TABLE-US-00002 TABLE 1 Sequences of CS and Hep17 synthetic peptides. CSP CD8+ T cell epitopes SI9 Rs 280-88 SYVPSAEQI Dominant IL10 Rs 58-67 IYNRNIVNRL Subdominant CD4+ T helper cell epitopes with overlapping CD8+ T cell epitopes SI16 Rs 280-95 SYVPSAEQILEFVKQI Dominant KD14 Rs 57-70 KIYNRNIVNRLLGD Dominant (nested dom. CD8+ T cell epitope) YK21 Rs 59-79 YNRNIVNRLLGDALNGKPEEK Subdominant (nested subdom. CD8+ T cell epitope) PyHEP17 CD8+ T cell epitopes (9-mer) L9A Rs 73-81 LRKINVALA Subdominant EN9 Rs 61-69 EEIVKLTKN Subdominant KV9 Rs 70-78 KKSLRKINV Subdominant IL9 Rs 76-84 INVALATAL Subdominant LL9 Rs 84-92 LSVVSAILL Subdominant CD4+ T cell epitope with nested CD8+ T cell epitopes (15-mer) EK15 Rs 61-75 EEIVKLTKNKKSLRK Dominant KL14 Rs 71-84 KSLRKINVALATAL Dominant Ref CSP: G. Del Giudice et al., Immunol Lett 25 (1990), pp. 59-63 E.D. Franke, Infect Immun 68 (2000), pp. 3403-3411 L. Renia, Proc Natl Acad Sci USA 88 (1991), pp. 7963-7967. W.R. Weiss, J Exp Med 171 (1990), pp. 763-773. Py HEP17 Y. Charoenvit,, Infect Immun 67 (1999), pp. 5604-5614. C. Dobano, Mol Immunol 44 (11) (2007), pp. 3037-3048.

[0243] Recombinant MSP1₁₉ protein. P. yoelii YM MSP1₁₉ (aa 1649-1757) was amplified by PCR using the forward primer 5'-CGTGGATCCATGGACGGCATGGATCTGCTG-3' and the reverse primer 5'-GATGAATTCGGAGCTGCTGCTGCAGAACACG-3' from pTRIP MSP1₄₂ and cloned into the glutathione 5-transferase (GST)-fusion protein expression vector pGEX-2T (Amersham Biosciences, Bucks, UK). Escherichia coli BL21 star (Invitrogen) were transformed with pGEX-2T MSP1₁₉ and growth and induction were performed according to the manufacturer's instructions (pGEX vectors, GST gene fusion system, Amersham). After induction of the expression of the protein in BL21, cells were harvested and lysed using BugBuster reagent (Novagen). Recombinant protein was purified by GST bind resin chromatography using GST bind purification kit (Novagen) as per manufacturer's instructions.

[0244] Measurement of Serum Antibody Responses.

[0245] Sera were collected 3 weeks after the last immunization for the assessment of MSP1₁₉-specific antibodies by enzyme-linked immunosorbent assay (ELISA). Recombinant GST- MSP1₁₉ fusion protein or GST control were adsorbed overnight at 4° C. to 96 well Nunc-Immuno Maxisorp plates (Fischer Scientific, Wohlen, Germany) at 2 μg/ml in PBS. After three washes with 0.05% Tween 20 in PBS, wells were blocked by the addition of 100 μl of PBS containing 10% of foetal bovine serum (FBS) at room temperature for 1 hour. Plates were washed three times with 0.05% Tween 20 in PBS and 100 μl of tenfold serial dilutions of serum were added to the wells. After incubation for 2 hours at room temperature, the wells were washed and 100 μl of peroxydase goat anti-mouse immunoglobulin (H+L) (Jackson Immuno Research) diluted 1/4000 in PBS 10% FBS was added to each well. After incubation for 1 hour at room temperature, wells were washed and 100 μl of tetramethylbenzidine substrate reagent (BD Pharmingen) was added to each well. The plates were incubated at room temperature for 30 min, and 100 μl of 1N H₂SO₄ was added to stop the reaction. The plates were read for optical density at 450 nm. The endpoint titer was calculated as the reciprocal of the last dilution eliciting twice the optical density of sera from non immunized mice.

REFERENCES

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Sequence CWU 1

1

90133DNAartificialoligonucleotide 1ggtaccggat ccgccaccat gaagaaatgc acc 33243DNAArtificialoligonucleotide 2agctcgagtc atcacaggct gttggacacg atgttgaaga tgc 43317PRTartificialsequence coding for the secretion signal of the calreticuline 3Met Leu Leu Ser Val Pro Leu Leu Leu Gly Leu Leu Gly Leu Ala Val 1 5 10 15 Ala 425DNAartificialprimer 4ggggattggt tttgacgttt ttgcg 25530DNAArtificialprimer 5aagcattaaa taaagcgaat acatccttat 3069PRTArtificialpeptide from Plasmodium yoelii cs protein 6Ser Tyr Val Pro Ser Ala Glu Gln Ile 1 5 710PRTArtificialpeptide from Plasmodium yoelii cs protein 7Ile Tyr Asn Arg Asn Ile Val Asn Arg Leu 1 5 10 816PRTArtificialpeptide from Plasmodium yoelii cs protein 8Ser Tyr Val Pro Ser Ala Glu Gln Ile Leu Glu Phe Val Lys Gln Ile 1 5 10 15 914PRTArtificialsynthetic peptide from Plasmodium yoelii CSP 9Lys Ile Tyr Asn Arg Asn Ile Val Asn Arg Leu Leu Gly Asp 1 5 10 1021PRTArtificialsynthetic peptide from Plasmodium yoelii CSP 10Tyr Asn Arg Asn Ile Val Asn Arg Leu Leu Gly Asp Ala Leu Asn Gly 1 5 10 15 Lys Pro Glu Glu Lys 20 119PRTArtificialsynthetic peptide from Plasmodium yoelii Hep17 protein 11Leu Arg Lys Ile Asn Val Ala Leu Ala 1 5 129PRTArtificialsynthetic peptide from Plasmodium yoelii Hep17 protein 12Glu Glu Ile Val Lys Leu Thr Lys Asn 1 5 139PRTArtificialsynthetic peptide from Plasmodium yoelii Hep17 protein 13Lys Lys Ser Leu Arg Lys Ile Asn Val 1 5 149PRTArtificialsynthetic peptide from Plasmodium yoelii Hep17 protein 14Ile Asn Val Ala Leu Ala Thr Ala Leu 1 5 159PRTArtificialsynthetic peptide from Plasmodium yoelii Hep17 protein 15Leu Ser Val Val Ser Ala Ile Leu Leu 1 5 1615PRTArtificialsynthetic peptide from Plasmodium yoelii Hep17 protein 16Glu Glu Ile Val Lys Leu Thr Lys Asn Lys Lys Ser Leu Arg Lys 1 5 10 15 1714PRTArtificialsynthetic peptide from Plasmodium yoelii Hep17 protein 17Lys Ser Leu Arg Lys Ile Asn Val Ala Leu Ala Thr Ala Leu 1 5 10 1830DNAArtificialprimer 18cgtggatcca tggacggcat ggatctgctg 301931DNAArtificialprimer 19gatgaattcg gagctgctgc tgcagaacac g 3120367PRTPlasmodium yoeliiMISC_FEATURE(1)..(367)CSP protein 20Met Lys Lys Cys Thr Ile Leu Val Val Ala Ser Leu Leu Leu Val Asp 1 5 10 15 Ser Leu Leu Pro Gly Tyr Gly Gln Asn Lys Ser Val Gln Ala Gln Arg 20 25 30 Asn Leu Asn Glu Leu Cys Tyr Asn Glu Glu Asn Asp Asn Lys Leu Tyr 35 40 45 His Val Leu Asn Ser Lys Asn Gly Lys Ile Tyr Asn Arg Asn Ile Val 50 55 60 Asn Arg Leu Leu Gly Asp Ala Leu Asn Gly Lys Pro Glu Glu Lys Lys 65 70 75 80 Asp Asp Pro Pro Lys Asp Gly Asn Lys Asp Asp Leu Pro Lys Glu Glu 85 90 95 Lys Lys Asp Asp Leu Pro Lys Glu Glu Lys Lys Asp Asp Pro Pro Lys 100 105 110 Asp Pro Lys Lys Asp Asp Pro Pro Lys Glu Ala Gln Asn Lys Leu Asn 115 120 125 Gln Pro Val Val Ala Asp Glu Asn Val Asp Gln Gly Pro Gly Ala Pro 130 135 140 Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly 145 150 155 160 Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly 165 170 175 Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro 180 185 190 Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly 195 200 205 Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly 210 215 220 Pro Gly Ala Pro Gln Glu Pro Pro Gln Gln Pro Pro Gln Gln Pro Pro 225 230 235 240 Gln Gln Pro Pro Gln Gln Pro Pro Gln Gln Pro Pro Gln Gln Pro Pro 245 250 255 Gln Gln Pro Arg Pro Gln Pro Asp Gly Asn Asn Asn Asn Asn Asn Asn 260 265 270 Asn Gly Asn Asn Asn Glu Asp Ser Tyr Val Pro Ser Ala Glu Gln Ile 275 280 285 Leu Glu Phe Val Lys Gln Ile Ser Ser Gln Leu Thr Glu Glu Trp Ser 290 295 300 Gln Cys Ser Val Thr Cys Gly Ser Gly Val Arg Val Arg Lys Arg Lys 305 310 315 320 Asn Val Asn Lys Gln Pro Glu Asn Leu Thr Leu Glu Asp Ile Asp Thr 325 330 335 Glu Ile Cys Lys Met Asp Lys Cys Ser Ser Ile Phe Asn Ile Val Ser 340 345 350 Asn Ser Leu Gly Phe Val Ile Leu Leu Val Leu Val Phe Phe Asn 355 360 365 21355PRTPlasmodium yoeliiMISC_FEATURE(1)..(355)CSP DGPI protein (deletion from residues GFVILLVLVFFN from CSP) 21Met Lys Lys Cys Thr Ile Leu Val Val Ala Ser Leu Leu Leu Val Asp 1 5 10 15 Ser Leu Leu Pro Gly Tyr Gly Gln Asn Lys Ser Val Gln Ala Gln Arg 20 25 30 Asn Leu Asn Glu Leu Cys Tyr Asn Glu Glu Asn Asp Asn Lys Leu Tyr 35 40 45 His Val Leu Asn Ser Lys Asn Gly Lys Ile Tyr Asn Arg Asn Ile Val 50 55 60 Asn Arg Leu Leu Gly Asp Ala Leu Asn Gly Lys Pro Glu Glu Lys Lys 65 70 75 80 Asp Asp Pro Pro Lys Asp Gly Asn Lys Asp Asp Leu Pro Lys Glu Glu 85 90 95 Lys Lys Asp Asp Leu Pro Lys Glu Glu Lys Lys Asp Asp Pro Pro Lys 100 105 110 Asp Pro Lys Lys Asp Asp Pro Pro Lys Glu Ala Gln Asn Lys Leu Asn 115 120 125 Gln Pro Val Val Ala Asp Glu Asn Val Asp Gln Gly Pro Gly Ala Pro 130 135 140 Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly 145 150 155 160 Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly 165 170 175 Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro 180 185 190 Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly 195 200 205 Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly 210 215 220 Pro Gly Ala Pro Gln Glu Pro Pro Gln Gln Pro Pro Gln Gln Pro Pro 225 230 235 240 Gln Gln Pro Pro Gln Gln Pro Pro Gln Gln Pro Pro Gln Gln Pro Pro 245 250 255 Gln Gln Pro Arg Pro Gln Pro Asp Gly Asn Asn Asn Asn Asn Asn Asn 260 265 270 Asn Gly Asn Asn Asn Glu Asp Ser Tyr Val Pro Ser Ala Glu Gln Ile 275 280 285 Leu Glu Phe Val Lys Gln Ile Ser Ser Gln Leu Thr Glu Glu Trp Ser 290 295 300 Gln Cys Ser Val Thr Cys Gly Ser Gly Val Arg Val Arg Lys Arg Lys 305 310 315 320 Asn Val Asn Lys Gln Pro Glu Asn Leu Thr Leu Glu Asp Ile Asp Thr 325 330 335 Glu Ile Cys Lys Met Asp Lys Cys Ser Ser Ile Phe Asn Ile Val Ser 340 345 350 Asn Ser Leu 355 22349PRTPlasmodium yoeliiMISC_FEATURE(1)..(349)CSP NTer protein (Deletion of residues 1-19 from CSP) 22Met Pro Gly Tyr Gly Gln Asn Lys Ser Val Gln Ala Gln Arg Asn Leu 1 5 10 15 Asn Glu Leu Cys Tyr Asn Glu Glu Asn Asp Asn Lys Leu Tyr His Val 20 25 30 Leu Asn Ser Lys Asn Gly Lys Ile Tyr Asn Arg Asn Ile Val Asn Arg 35 40 45 Leu Leu Gly Asp Ala Leu Asn Gly Lys Pro Glu Glu Lys Lys Asp Asp 50 55 60 Pro Pro Lys Asp Gly Asn Lys Asp Asp Leu Pro Lys Glu Glu Lys Lys 65 70 75 80 Asp Asp Leu Pro Lys Glu Glu Lys Lys Asp Asp Pro Pro Lys Asp Pro 85 90 95 Lys Lys Asp Asp Pro Pro Lys Glu Ala Gln Asn Lys Leu Asn Gln Pro 100 105 110 Val Val Ala Asp Glu Asn Val Asp Gln Gly Pro Gly Ala Pro Gln Gly 115 120 125 Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro 130 135 140 Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly 145 150 155 160 Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly 165 170 175 Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro 180 185 190 Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly 195 200 205 Ala Pro Gln Glu Pro Pro Gln Gln Pro Pro Gln Gln Pro Pro Gln Gln 210 215 220 Pro Pro Gln Gln Pro Pro Gln Gln Pro Pro Gln Gln Pro Pro Gln Gln 225 230 235 240 Pro Arg Pro Gln Pro Asp Gly Asn Asn Asn Asn Asn Asn Asn Asn Gly 245 250 255 Asn Asn Asn Glu Asp Ser Tyr Val Pro Ser Ala Glu Gln Ile Leu Glu 260 265 270 Phe Val Lys Gln Ile Ser Ser Gln Leu Thr Glu Glu Trp Ser Gln Cys 275 280 285 Ser Val Thr Cys Gly Ser Gly Val Arg Val Arg Lys Arg Lys Asn Val 290 295 300 Asn Lys Gln Pro Glu Asn Leu Thr Leu Glu Asp Ile Asp Thr Glu Ile 305 310 315 320 Cys Lys Met Asp Lys Cys Ser Ser Ile Phe Asn Ile Val Ser Asn Ser 325 330 335 Leu Gly Phe Val Ile Leu Leu Val Leu Val Phe Phe Asn 340 345 23397PRTPlasmodium falciparumMISC_FEATURE(1)..(397)CSP protein 23Met Met Arg Lys Leu Ala Ile Leu Ser Val Ser Ser Phe Leu Phe Val 1 5 10 15 Glu Ala Leu Phe Gln Glu Tyr Gln Cys Tyr Gly Ser Ser Ser Asn Thr 20 25 30 Arg Val Leu Asn Glu Leu Asn Tyr Asp Asn Ala Gly Thr Asn Leu Tyr 35 40 45 Asn Glu Leu Glu Met Asn Tyr Tyr Gly Lys Gln Glu Asn Trp Tyr Ser 50 55 60 Leu Lys Lys Asn Ser Arg Ser Leu Gly Glu Asn Asp Asp Gly Asn Asn 65 70 75 80 Glu Asp Asn Glu Lys Leu Arg Lys Pro Lys His Lys Lys Leu Lys Gln 85 90 95 Pro Ala Asp Gly Asn Pro Asp Pro Asn Ala Asn Pro Asn Val Asp Pro 100 105 110 Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn Val Asp Pro 115 120 125 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 130 135 140 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 145 150 155 160 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 165 170 175 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 180 185 190 Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro 195 200 205 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 210 215 220 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 225 230 235 240 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 245 250 255 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 260 265 270 Asn Lys Asn Asn Gln Gly Asn Gly Gln Gly His Asn Met Pro Asn Asp 275 280 285 Pro Asn Arg Asn Val Asp Glu Asn Ala Asn Ala Asn Ser Ala Val Lys 290 295 300 Asn Asn Asn Asn Glu Glu Pro Ser Asp Lys His Ile Lys Glu Tyr Leu 305 310 315 320 Asn Lys Ile Gln Asn Ser Leu Ser Thr Glu Trp Ser Pro Cys Ser Val 325 330 335 Thr Cys Gly Asn Gly Ile Gln Val Arg Ile Lys Pro Gly Ser Ala Asn 340 345 350 Lys Pro Lys Asp Glu Leu Asp Tyr Ala Asn Asp Ile Glu Lys Lys Ile 355 360 365 Cys Lys Met Glu Lys Cys Ser Ser Val Phe Asn Val Val Asn Ser Ser 370 375 380 Ile Gly Leu Ile Met Val Leu Ser Phe Leu Phe Leu Asn 385 390 395 24368PRTPlasmodium falciparumMISC_FEATURE(1)..(368)CSP DGPI protein 24Met Met Arg Lys Leu Ala Ile Leu Ser Val Ser Ser Phe Leu Phe Val 1 5 10 15 Glu Ala Leu Phe Gln Glu Tyr Gln Cys Tyr Gly Ser Ser Ser Asn Thr 20 25 30 Arg Val Leu Asn Glu Leu Asn Tyr Asp Asn Ala Gly Thr Asn Leu Tyr 35 40 45 Asn Glu Leu Glu Met Asn Tyr Tyr Gly Lys Gln Glu Asn Trp Tyr Ser 50 55 60 Leu Lys Lys Asn Ser Arg Ser Leu Gly Glu Asn Asp Asp Gly Asn Asn 65 70 75 80 Glu Asp Asn Glu Lys Leu Arg Lys Pro Lys His Lys Lys Leu Lys Gln 85 90 95 Pro Ala Asp Gly Asn Pro Asp Pro Asn Ala Asn Pro Asn Val Asp Pro 100 105 110 Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn Val Asp Pro 115 120 125 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 130 135 140 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 145 150 155 160 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 165 170 175 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 180 185 190 Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro 195 200 205 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 210 215 220 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 225 230 235 240 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 245 250 255 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 260 265 270 Asn Lys Asn Asn Gln Gly Asn Gly Gln Gly His Asn Met Pro Asn Asp 275 280 285 Pro Asn Arg Asn Val Asp Glu Asn Ala Asn Ala Asn Ser Ala Val Lys 290 295 300 Asn Asn Asn Asn Glu Glu Pro Ser Asp Lys His Ile Lys Glu Tyr Leu 305 310 315 320 Asn Lys Ile Gln Asn Ser Leu Ser Thr Glu Trp Ser Pro Cys Ser Val 325 330 335 Thr Cys Gly Asn Gly Ile Gln Val Arg Ile Lys Pro Gly Ser Ala Asn 340 345 350 Lys Pro Lys Asp Glu Leu Asp Tyr Ala Asn Asp Ile Glu Lys Lys Ile 355 360 365 25378PRTPlasmodium falciparumMISC_FEATURE(1)..(378)CSP NTer protein 25Met Gln Glu Tyr Gln Cys Tyr Gly Ser Ser Ser Asn Thr Arg Val Leu 1 5 10 15 Asn Glu Leu Asn Tyr Asp Asn Ala Gly Thr Asn Leu Tyr Asn Glu Leu 20 25 30 Glu Met Asn Tyr Tyr Gly Lys Gln Glu Asn

Trp Tyr Ser Leu Lys Lys 35 40 45 Asn Ser Arg Ser Leu Gly Glu Asn Asp Asp Gly Asn Asn Glu Asp Asn 50 55 60 Glu Lys Leu Arg Lys Pro Lys His Lys Lys Leu Lys Gln Pro Ala Asp 65 70 75 80 Gly Asn Pro Asp Pro Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn 85 90 95 Pro Asn Val Asp Pro Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn 100 105 110 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 115 120 125 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 130 135 140 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 145 150 155 160 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 165 170 175 Pro Asn Val Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 180 185 190 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 195 200 205 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 210 215 220 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 225 230 235 240 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Lys Asn 245 250 255 Asn Gln Gly Asn Gly Gln Gly His Asn Met Pro Asn Asp Pro Asn Arg 260 265 270 Asn Val Asp Glu Asn Ala Asn Ala Asn Ser Ala Val Lys Asn Asn Asn 275 280 285 Asn Glu Glu Pro Ser Asp Lys His Ile Lys Glu Tyr Leu Asn Lys Ile 290 295 300 Gln Asn Ser Leu Ser Thr Glu Trp Ser Pro Cys Ser Val Thr Cys Gly 305 310 315 320 Asn Gly Ile Gln Val Arg Ile Lys Pro Gly Ser Ala Asn Lys Pro Lys 325 330 335 Asp Glu Leu Asp Tyr Ala Asn Asp Ile Glu Lys Lys Ile Cys Lys Met 340 345 350 Glu Lys Cys Ser Ser Val Phe Asn Val Val Asn Ser Ser Ile Gly Leu 355 360 365 Ile Met Val Leu Ser Phe Leu Phe Leu Asn 370 375 26329PRTPlasmodium bergheiMISC_FEATURE(1)..(329)CSP protein 26Met Lys Lys Cys Thr Ile Leu Val Val Ala Ser Leu Leu Leu Val Asn 1 5 10 15 Ser Leu Leu Pro Gly Tyr Gly Gln Asn Lys Ile Ile Gln Ala Gln Arg 20 25 30 Asn Leu Asn Glu Leu Cys Tyr Asn Glu Gly Asn Asp Asn Lys Leu Tyr 35 40 45 His Val Leu Asn Ser Lys Asn Gly Lys Ile Tyr Asn Arg Asn Thr Val 50 55 60 Asn Arg Leu Leu Ala Asp Ala Pro Glu Gly Lys Lys Asn Glu Lys Lys 65 70 75 80 Asn Glu Lys Ile Glu Arg Asn Asn Lys Leu Lys Pro Pro Pro Asn Pro 85 90 95 Asn Asp Pro Pro Pro Pro Asn Pro Asn Asp Pro Pro Pro Pro Asn Pro 100 105 110 Asn Asp Pro Pro Pro Pro Asn Pro Asn Asp Pro Ala Pro Pro Asn Ala 115 120 125 Asn Asp Pro Ala Pro Pro Asn Ala Asn Asp Pro Ala Pro Pro Asn Ala 130 135 140 Asn Asp Pro Ala Pro Pro Asn Ala Asn Asp Pro Ala Pro Pro Asn Ala 145 150 155 160 Asn Asp Pro Ala Pro Pro Asn Ala Asn Asp Pro Ala Pro Pro Asn Ala 165 170 175 Asn Asp Pro Pro Pro Pro Asn Pro Asn Asp Pro Ala Pro Pro Gln Gly 180 185 190 Asn Asn Asn Pro Gln Pro Gln Pro Arg Pro Gln Pro Gln Pro Gln Pro 195 200 205 Gln Pro Gln Pro Gln Pro Gln Pro Gln Pro Gln Pro Arg Pro Gln Pro 210 215 220 Gln Pro Gln Pro Gly Gly Asn Asn Asn Asn Lys Asn Asn Asn Asn Asp 225 230 235 240 Asp Ser Tyr Ile Pro Ser Ala Glu Lys Ile Leu Glu Phe Val Lys Gln 245 250 255 Ile Arg Asp Ser Ile Thr Glu Glu Trp Ser Gln Cys Asn Val Thr Cys 260 265 270 Gly Ser Gly Ile Arg Val Arg Lys Arg Lys Gly Ser Asn Lys Lys Ala 275 280 285 Glu Asp Leu Thr Leu Glu Asp Ile Asp Thr Glu Ile Cys Lys Met Asp 290 295 300 Lys Cys Ser Ser Ile Phe Asn Ile Val Ser Asn Ser Leu Gly Phe Val 305 310 315 320 Ile Leu Leu Val Leu Val Phe Phe Asn 325 27429PRTPlasmodium malariaeMISC_FEATURE(1)..(429)CSP protein 27Met Lys Lys Leu Ser Val Leu Ala Ile Ser Ser Phe Leu Ile Val Asp 1 5 10 15 Phe Leu Phe Pro Gly Tyr His His Asn Ser Asn Ser Thr Lys Ser Arg 20 25 30 Asn Leu Ser Glu Leu Cys Tyr Asn Asn Val Asp Thr Lys Leu Phe Asn 35 40 45 Glu Leu Glu Val Arg Tyr Ser Thr Asn Gln Asp His Phe Tyr Asn Tyr 50 55 60 Asn Lys Thr Ile Arg Leu Leu Asn Glu Asn Asn Asn Glu Lys Asp Gly 65 70 75 80 Asn Val Thr Asn Glu Arg Lys Lys Lys Pro Thr Lys Ala Val Glu Asn 85 90 95 Lys Leu Lys Gln Pro Pro Gly Asp Asp Asp Gly Ala Gly Asn Asp Ala 100 105 110 Gly Asn Asp Ala Gly Asn Asp Ala Gly Asn Ala Ala Gly Asn Ala Ala 115 120 125 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 130 135 140 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 145 150 155 160 Gly Asn Asp Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 165 170 175 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Asp Ala Gly Asn Ala Ala 180 185 190 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 195 200 205 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 210 215 220 Gly Asn Ala Ala Gly Asn Asp Ala Gly Asn Ala Ala Gly Asn Ala Ala 225 230 235 240 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 245 250 255 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 260 265 270 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 275 280 285 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 290 295 300 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Glu Lys Ala Lys Asn Lys 305 310 315 320 Asp Asn Lys Val Asp Ala Asn Thr Asn Lys Lys Asp Asn Gln Glu Glu 325 330 335 Asn Asn Asp Ser Ser Asn Gly Pro Ser Glu Glu His Ile Lys Asn Tyr 340 345 350 Leu Glu Ser Ile Arg Asn Ser Ile Thr Glu Glu Trp Ser Pro Cys Ser 355 360 365 Val Thr Cys Gly Ser Gly Ile Arg Ala Arg Arg Lys Val Gly Ala Lys 370 375 380 Asn Lys Lys Pro Ala Glu Leu Val Leu Ser Asp Leu Glu Thr Glu Ile 385 390 395 400 Cys Ser Leu Asp Lys Cys Ser Ser Ile Phe Asn Val Val Ser Asn Ser 405 410 415 Leu Gly Ile Val Leu Val Leu Val Leu Ile Leu Phe His 420 425 28344PRTPlasmodium coatneyiMISC_FEATURE(1)..(344)CSP protein 28Met Lys Asn Phe Ile Leu Leu Ala Val Ser Ser Ile Leu Leu Val Asp 1 5 10 15 Leu Phe Pro Thr His Phe Gly His Asn Val Asp Leu Ser Arg Ala Ile 20 25 30 Asn Leu Asn Gly Val Ser Phe Asn Asn Val Asp Thr Ser Leu Leu Gly 35 40 45 Ala Ala Gln Val Arg Gln Ser Ala Ser Arg Gly Arg Gly Leu Gly Glu 50 55 60 Lys Pro Lys Lys Lys Ala Glu Lys Lys Glu Glu Glu Pro Lys Lys Pro 65 70 75 80 Asn Glu Asn Lys Cys Ser Pro Leu Lys Gln Pro Val Asp Gly Ala Arg 85 90 95 Asp Gly Pro Ala Pro Ala Ala Asp Gly Ala Arg Asp Gly Pro Ala Pro 100 105 110 Ala Ala Asp Gly Ala Arg Asp Gly Pro Ala Pro Ala Ala Asp Gly Ala 115 120 125 Arg Asp Gly Pro Ala Pro Ala Ala Asp Gly Ala Arg Asp Gly Pro Ala 130 135 140 Pro Ala Ala Asp Gly Ala Arg Asp Gly Pro Ala Pro Ala Ala Asp Gly 145 150 155 160 Ala Arg Asp Gly Pro Ala Pro Ala Ala Asp Gly Ala Arg Asp Gly Pro 165 170 175 Ala Pro Pro Ala Asp Gly Ala Arg Asp Gly Pro Ala Pro Pro Ala Ala 180 185 190 Asp Gly Ala Arg Asp Gly Pro Ala Pro Pro Ala Ala Asp Gly Ala Arg 195 200 205 Asp Gly Pro Ala Pro Pro Ala Gly Gln Gly Gly Gly Asn Ala Ala Gly 210 215 220 Gln Ala Gln Gly Gly Gly Asn Ala Gly Asn Lys Lys Ala Gly Asp Ala 225 230 235 240 Ala Gly Asn Ala Gly Ala Ala Lys Gly Gln Gly Gln Asn Asn Glu Gly 245 250 255 Ala Asn Val Pro Asn Glu Lys Val Val Asn Asp Tyr Leu Gln Lys Ile 260 265 270 Arg Ser Thr Val Thr Thr Glu Trp Thr Pro Cys Ser Val Thr Cys Gly 275 280 285 Asn Gly Val Arg Leu Arg Arg Lys Ala His Ala Glu Lys Lys Lys Pro 290 295 300 Glu Asp Leu Thr Met Asp Asp Leu Asp Val Glu Val Cys Ala Met Asp 305 310 315 320 Lys Cys Ala Gly Ile Phe Asn Phe Val Ser Asn Ser Leu Gly Leu Val 325 330 335 Ile Leu Leu Val Leu Ala Phe Asn 340 29347PRTPlasmodium knowlesiMISC_FEATURE(1)..(347)CSP protein 29Met Arg Asn Phe Ile Leu Leu Ala Val Ser Ser Ile Leu Leu Val Asp 1 5 10 15 Leu Phe Pro Thr His Phe Glu His Asn Val Asp Leu Ser Arg Ala Ile 20 25 30 Asn Val Asn Gly Val Ser Phe Asn Asn Val Asp Thr Ser Ser Leu Gly 35 40 45 Ala Ala Gln Val Arg Gln Ser Ala Ser Arg Gly Arg Gly Leu Gly Glu 50 55 60 Lys Arg Lys Glu Gly Ala Asp Lys Glu Lys Lys Lys Glu Lys Glu Glu 65 70 75 80 Glu Pro Lys Lys Pro Asn Glu Asn Lys Leu Lys Gln Pro Asp Gln Ala 85 90 95 Ala Pro Gly Ala Gly Gly Glu Gln Pro Ala Pro Gly Ala Gly Gly Glu 100 105 110 Gln Pro Ala Pro Gly Ala Gly Gly Glu Arg Pro Ala Pro Gly Ala Gly 115 120 125 Gly Glu Gln Pro Ala Pro Gly Ala Gly Gly Glu Gln Pro Ala Pro Gly 130 135 140 Ala Gly Gly Glu Arg Pro Ala Pro Gly Ala Gly Gly Glu Gln Pro Ala 145 150 155 160 Pro Gly Ala Gly Gly Glu Gln Pro Ala Pro Gly Ala Gly Gly Glu Gln 165 170 175 Pro Ala Pro Gly Ala Gly Gly Glu Gln Pro Ala Pro Gly Ala Gly Gly 180 185 190 Glu Arg Pro Ala Pro Gly Ala Gly Gly Glu Arg Pro Ala Pro Gly Ala 195 200 205 Gly Gly Glu Gln Pro Ala Pro Gly Ala Gly Gly Glu Gln Pro Ala Pro 210 215 220 Ala Pro Arg Arg Glu Gln Pro Ala Pro Gly Pro Gly Ala Gly Asp Gly 225 230 235 240 Ala Arg Gly Gly Asn Ala Gly Ala Gly Lys Gly Gln Gly Gln Asn Asn 245 250 255 Gln Gly Ala Asn Val Pro Asn Glu Lys Val Val Asn Asp Tyr Leu His 260 265 270 Lys Ile Arg Ser Ser Val Thr Thr Glu Trp Thr Pro Cys Ser Val Thr 275 280 285 Cys Gly Asn Gly Val Arg Ile Arg Arg Arg Gln Asn Ala Gly Asn Lys 290 295 300 Lys Ala Glu Asp Leu Thr Met Asp Asp Leu Glu Val Glu Ala Cys Val 305 310 315 320 Met Asp Lys Cys Ala Gly Ile Phe Asn Val Val Ser Asn Ser Leu Gly 325 330 335 Leu Val Ile Leu Leu Val Leu Ala Leu Phe Asn 340 345 30388PRTPlasmodium reichenowi 30Met Met Arg Lys Leu Ala Ile Leu Ser Val Ser Ser Phe Leu Phe Val 1 5 10 15 Glu Ala Leu Phe Gln Glu Tyr Gln Cys Tyr Gly Ser Ser Ser Asn Thr 20 25 30 Arg Val Leu Asn Glu Leu Asn Tyr Asp Asn Ala Gly Thr Asn Leu Tyr 35 40 45 Asn Glu Leu Glu Met Asn Tyr Tyr Gly Lys Gln Glu Asn Trp Tyr Ser 50 55 60 Leu Lys Lys Asn Ser Arg Ser Leu Gly Glu Asn Asp Asp Ala Asp Asn 65 70 75 80 Gly Asp Ala Asp Asn Gly Asp Glu Gly Ile Asp Glu Asn Arg Arg His 85 90 95 Arg Asn Lys Glu Gly Lys Glu Lys Leu Lys Lys Pro Lys His Asn Lys 100 105 110 Leu Lys Gln Pro Gly Asn Asp Asn Val Asp Pro Asn Ala Asn Pro Asn 115 120 125 Val Asp Pro Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn 130 135 140 Val Asp Pro Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn 145 150 155 160 Val Asn Pro Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn 165 170 175 Val Asn Pro Asn Ala Asn Pro Asn Val Asn Pro Asn Ala Asn Pro Asn 180 185 190 Val Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 195 200 205 Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 210 215 220 Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 225 230 235 240 Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 245 250 255 Ala Asn Pro Asn Ala Asn Pro Asn Arg Asn Asn Glu Ala Asn Gly Gln 260 265 270 Gly His Asn Lys Pro Asn Asp Gln Asn Arg Asn Val Asn Glu Asn Ala 275 280 285 Asn Ala Asn Asn Ala Gly Arg Asn Asn Asn Asn Glu Glu Pro Ser Asp 290 295 300 Lys His Ile Glu Glu Phe Leu Lys Gln Ile Gln Asn Asn Leu Ser Thr 305 310 315 320 Glu Trp Ser Pro Cys Ser Val Thr Cys Gly Asn Gly Ile Gln Val Arg 325 330 335 Ile Lys Pro Gly Ser Ala Gly Lys Pro Lys Asp Gln Leu Asp Tyr Glu 340 345 350 Asn Asp Leu Glu Lys Lys Ile Cys Lys Met Glu Lys Cys Ser Ser Val 355 360 365 Phe Asn Val Val Asn Ser Ser Ile Gly Leu Ile Met Val Leu Ser Phe 370 375 380 Leu Phe Leu Asn 385 31388PRTPlasmodium gallinaceum 31Met Lys Lys Leu Ala Ile Leu Ser Ala Ser Ser Phe Leu Phe Ala Asp 1 5 10 15 Phe Leu Phe Gln Glu Tyr Gln His Asn Gly Asn Tyr Lys Asn Phe Arg 20 25 30 Leu Leu Asn Glu Val Cys Tyr Asn Asn Met Asn Ile Gln Leu Tyr Asn 35 40 45 Glu Leu Glu Met Glu Asn Tyr Met Ser Asn Thr Tyr Phe Tyr Asn Asn 50 55 60 Lys Lys Thr Ile Arg Leu Leu Gly Glu Asn Asp Asn Glu Ala Asn Val 65 70 75 80 Asn Arg Ala Asn Asn

Asn Val Ala Asn Asp Asn Arg Ala Asn Gly Asn 85 90 95 Arg Gly Asn Val Asn Arg Ala Asn Asp Arg Asn Ile Pro Tyr Phe Arg 100 105 110 Glu Asn Val Val Asn Leu Asn Gln Pro Val Gly Gly Asn Gly Gly Val 115 120 125 Gln Pro Ala Gly Gly Asn Gly Gly Val Gln Pro Ala Gly Gly Asn Gly 130 135 140 Gly Val Gln Pro Ala Gly Gly Asn Gly Gly Val Gln Pro Ala Gly Gly 145 150 155 160 Asn Gly Gly Val Gln Pro Ala Gly Gly Asn Gly Gly Val Gln Pro Ala 165 170 175 Gly Gly Asn Gly Gly Val Gln Pro Ala Gly Gly Asn Gly Gly Ala Gln 180 185 190 Pro Val Ala Ala Gly Gly Gly Ala Gln Pro Val Val Ala Asp Gly Gly 195 200 205 Val Gln Pro Leu Arg Gln Glu Gly Asp Ala Glu Glu Asp Gly Gly Asn 210 215 220 Gly Gly Ala Gln Pro Ala Gly Gly Asn Gly Gly Ala Gln Pro Ala Gly 225 230 235 240 Gly Asn Gly Gly Ala Gln Pro Ala Gly Gly Asn Gly Gly Ala Gln Pro 245 250 255 Ala Gly Gly Asn Gly Gly Ala Gln Pro Ala Gly Gly Asn Asp Ala Ala 260 265 270 Lys Pro Asp Gly Gly Asn Asp Asp Asp Lys Pro Glu Gly Gly Asp Glu 275 280 285 Lys Ser Glu Glu Glu Lys Glu Asp Glu Pro Ile Pro Asp Pro Thr Gln 290 295 300 Glu Glu Ile Asp Lys Tyr Leu Lys Ser Ile Leu Gly Asn Val Thr Ser 305 310 315 320 Glu Trp Thr Asn Cys Asn Val Thr Cys Gly Lys Gly Ile Gln Ala Lys 325 330 335 Ile Lys Ser Thr Ser Ala Asn Lys Lys Arg Glu Glu Ile Thr Pro Asn 340 345 350 Asp Val Glu Val Lys Ile Cys Glu Leu Glu Arg Cys Ser Phe Ser Ile 355 360 365 Phe Asn Val Ile Ser Asn Ser Leu Gly Leu Ala Ile Ile Leu Thr Phe 370 375 380 Leu Phe Phe Tyr 385 32258PRTConsensus 32Met Lys Lys Ile Leu Ser Val Ser Ser Ile Leu Leu Val Asp Ala Leu 1 5 10 15 Gln Tyr Asn Leu Ser Arg Asn Leu Asn Glu Leu Tyr Asn Tyr Asn Glu 20 25 30 Leu Glu Met His Val Gly Ala Asn Ser Arg Asn Gly Asn Asp Ala Asp 35 40 45 Glu Lys Glu Lys Lys Pro Asn Asn Lys Leu Pro Asn Ala Pro Asn Asp 50 55 60 Pro Ala Pro Asn Pro Ala Ala Gly Ala Ala Ala Ala Asn Asn Ala Pro 65 70 75 80 Ala Ala Asn Ala Pro Ala Pro Asn Ala Gly Asn Ala Pro Asn Ala Gly 85 90 95 Gly Ala Pro Asn Ala Asn Gly Ala Asn Pro Asn Ala Gly Ala Ala Pro 100 105 110 Pro Ala Gly Ala Asn Ala Pro Asn Ala Gly Pro Asn Ala Ala Gly Pro 115 120 125 Ala Gly Ala Ala Pro Asn Ala Pro Ala Ala Asn Gly Asn Ala Asn Pro 130 135 140 Asn Ala Pro Asn Ala Pro Asn Ala Pro Asn Ala Asn Pro Asn Ala Pro 145 150 155 160 Asn Asn Ala Gly Gln Asp Asn Ala Asn Asn Lys Asn Asn Gly Asn Asn 165 170 175 Glu Ser Val Pro Ser Glu Lys Ile Glu Tyr Leu Lys Ile Arg Ser Leu 180 185 190 Thr Thr Glu Trp Ser Pro Cys Ser Val Thr Cys Gly Asn Gly Ile Arg 195 200 205 Val Arg Arg Lys Gly Ser Ala Asn Lys Lys Glu Asp Leu Thr Leu Asp 210 215 220 Asp Leu Glu Glu Ile Cys Lys Met Asp Lys Cys Ser Ser Ile Phe Asn 225 230 235 240 Val Val Ser Asn Ser Leu Gly Leu Val Ile Leu Leu Val Leu Leu Phe 245 250 255 Phe Asn 334024DNAArtificialPLASMID pTRIP-DeltaU3-CMVeGFP 33tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca 60cacaaggcta cttccctgat tagcagaact acacaccagg gccagggatc agatatccac 120tgacctttgg atggtgctac aagctagtac cagttgagcc agagaagtta gaagaagcca 180acaaaggaga gaacaccagc ttgttacaac ctgtgagcct gcatgggatg gatgacccgg 240agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac ggtggcccga 300gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa gggactttcc 360gctgggggac tttccaggga ggcgtggcct gggcgggact ggggagtggc gagccctcag 420atcctgcata taagcagctg ctttttgcct gtactgggtc tctctggtta gaccagatct 480gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 540cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 600tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa 660agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac 720ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 780gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg 840ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg 900ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg 960ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag 1020atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga 1080caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca 1140gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg 1200aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa 1260agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt 1320tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca 1380gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc 1440aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg 1500ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac 1560tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga 1620tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa 1680tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg 1740aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata 1800taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac 1860tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc 1920caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca 1980gagacagatc cattcgatta gtgaacggat ctcgacggta tcgccgaatt cacaaatggc 2040agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag 2100aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa 2160aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggg gcgataagct 2220tgggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 2280ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 2340cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 2400tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 2460tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 2520atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 2580gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 2640caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 2700ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc 2760gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccgact ctagaggatc 2820cccaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat 2880cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga 2940gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc 3000cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta 3060ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca 3120ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt 3180cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg 3240caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc 3300cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg 3360cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct 3420gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa 3480gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga 3540cgagctgtac aagtaaagcg gccggactct agctcgagac ctagaaaaac atggagcaat 3600cacaagtagc aatacagcag ctaccaatgc tgattgtgcc tggctagaag cacaagagga 3660ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga cttacaaggc 3720agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc 3780ccaacgaaga caagatcgtc gagagatgct gcatataagc agctgctttt tgcttgtact 3840gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 3900ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 3960tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 4020agtg 4024344927DNAArtificialplasmid pTRIP-deltaU3-CMV-MSP142 CO-WPRE 34tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca 60cacaaggcta cttccctgat tagcagaact acacaccagg gccagggatc agatatccac 120tgacctttgg atggtgctac aagctagtac cagttgagcc agagaagtta gaagaagcca 180acaaaggaga gaacaccagc ttgttacaac ctgtgagcct gcatgggatg gatgacccgg 240agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac ggtggcccga 300gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa gggactttcc 360gctgggggac tttccaggga ggcgtggcct gggcgggact ggggagtggc gagccctcag 420atcctgcata taagcagctg ctttttgcct gtactgggtc tctctggtta gaccagatct 480gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 540cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 600tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa 660agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac 720ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 780gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg 840ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg 900ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg 960ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag 1020atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga 1080caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca 1140gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg 1200aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa 1260agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt 1320tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca 1380gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc 1440aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg 1500ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac 1560tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga 1620tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa 1680tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg 1740aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata 1800taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac 1860tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc 1920caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca 1980gagacagatc cattcgatta gtgaacggat ctcgacggta tcgccgaatt cacaaatggc 2040agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag 2100aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa 2160aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc tgatacgcgt 2220ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 2280ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 2340ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 2400tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 2460tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 2520cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 2580ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 2640aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 2700taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc 2760ctggagacgc catccacgct gttttgacct ccatagaaga caccgcgatc ggatccgcca 2820ccatgctgct gtccgtgccc ctgctgctgg gcctgctggg actggccgtg gccgctcccg 2880agaaggacat cctgagcgag ttcaccaacg agagcctgta cgtgtacaca aagagactgg 2940gcagcaccta caagagcctg aagaaacaca tgctgcggga gttcagcacc atcaaagaag 3000atatgaccaa cggcctgaac aacaagagcc agaagcggaa cgacttcctg gaggtgctgt 3060cccacgagct ggacctgttc aaggacctga gcaccaataa gtacgtgatc cggaacccct 3120accagctgct ggacaacgac aagaaggaca agcagatcgt caacctgaag tacgccacca 3180agggcatcaa cgaggatatc gagacaacca ccgacggcat caagttcttc aacaagatgg 3240tggagctgta caacacccag ctggccgccg tgaaggagca gatcgccacc atcgaggccg 3300agacaaacga cacaaacaag gaggagaaga agaagtacat ccccatcctg gaggacctga 3360agggcctgta cgagacagtg attggccagg ccgaggagta cagcgaggag ctgcagaaca 3420gactggataa ctacaagaac gagaaggccg agttcgagat cctgaccaag aacctggaga 3480agtacatcca gatcgacgag aagctggacg agttcgtgga gcacgccgag aacaacaagc 3540atatcgcctc tatcgccctg aacaacctga ataagagcgg cctggtggga gagggcgaga 3600gcaaaaagat cctggctaag atgctgaaca tggacggcat ggatctgctg ggcgtggacc 3660ccaagcacgt gtgcgtggac accagagaca tccccaagaa cgccggctgc ttcagggacg 3720acaacggcac cgaggagtgg agatgtctgc tgggctacaa gaagggcgag ggcaacacct 3780gcgtggagaa caataacccc acctgcgaca tcaacaacgg cggctgcgac cccaccgcca 3840gctgccagaa cgccgagagc accgagaact ccaagaagat catctgcacc tgcaaggagc 3900ccacccccaa cgcctactac gagggcgtgt tctgcagcag cagctccttc atgggctgat 3960gactcgagct caagcttcga attcccgata atcaacctct ggattacaaa atttgtgaaa 4020gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa 4080tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat 4140cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt 4200gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc 4260tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc 4320ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg 4380ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga 4440cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc 4500tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc 4560tttgggccgc ctccccgcgt cgacgcgtga attcggtacc tttaagacca atgacttaca 4620aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc 4680actcccaacg aagacaagat cgtcgagaga tgctgcatat aagcagctgc tttttgcttg 4740tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa 4800cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct 4860gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc 4920tagcagt 4927351140DNAArtificialPlasmid pTRIP-deltaU3-CMV-MSP142 CO-WPRE 35atg ctg ctg tcc gtg ccc ctg ctg ctg ggc ctg ctg gga ctg gcc gtg 48Met Leu Leu Ser Val Pro Leu Leu Leu Gly Leu Leu Gly Leu Ala Val 1 5 10 15 gcc gct ccc gag aag gac atc ctg agc gag ttc acc aac gag agc ctg 96Ala Ala Pro Glu Lys Asp Ile Leu Ser Glu Phe Thr Asn Glu Ser Leu 20 25 30 tac gtg tac aca aag aga ctg ggc agc acc tac aag agc ctg aag aaa 144Tyr Val Tyr Thr Lys Arg Leu Gly Ser Thr Tyr Lys Ser Leu Lys Lys 35 40 45 cac atg ctg cgg gag ttc agc acc atc aaa gaa gat atg acc aac ggc 192His Met Leu Arg Glu Phe Ser Thr Ile Lys Glu Asp Met Thr Asn Gly 50 55 60 ctg aac aac aag agc cag aag cgg aac gac ttc ctg gag gtg ctg tcc 240Leu Asn Asn Lys Ser Gln Lys Arg Asn Asp Phe Leu Glu Val Leu Ser 65 70 75 80 cac gag ctg gac ctg ttc aag gac ctg agc acc aat aag tac gtg atc 288His Glu Leu Asp Leu Phe Lys Asp Leu Ser Thr Asn Lys Tyr Val Ile 85 90 95 cgg aac ccc tac cag ctg ctg gac aac gac aag aag gac aag cag atc 336Arg Asn Pro Tyr Gln Leu Leu Asp Asn Asp Lys Lys Asp Lys Gln Ile 100 105 110 gtc aac ctg aag tac gcc acc aag ggc atc aac gag gat atc gag aca 384Val Asn Leu Lys Tyr Ala Thr Lys Gly Ile Asn Glu Asp Ile Glu Thr 115 120 125 acc acc gac ggc atc aag ttc ttc aac aag atg gtg gag ctg tac aac 432Thr Thr Asp Gly Ile Lys Phe Phe Asn Lys Met Val Glu Leu Tyr Asn 130 135 140 acc cag ctg gcc gcc gtg aag gag cag atc gcc acc atc gag gcc gag 480Thr Gln Leu Ala Ala Val Lys Glu Gln Ile Ala Thr Ile Glu Ala Glu 145 150 155 160 aca aac gac aca aac aag gag gag aag aag aag tac atc ccc atc ctg 528Thr Asn Asp Thr Asn Lys Glu Glu Lys Lys Lys Tyr Ile Pro Ile Leu 165 170 175 gag gac ctg aag ggc ctg tac gag aca gtg att ggc cag gcc gag gag 576Glu Asp Leu Lys Gly Leu Tyr Glu Thr Val Ile Gly Gln Ala Glu Glu 180 185 190

tac agc gag gag ctg cag aac aga ctg gat aac tac aag aac gag aag 624Tyr Ser Glu Glu Leu Gln Asn Arg Leu Asp Asn Tyr Lys Asn Glu Lys 195 200 205 gcc gag ttc gag atc ctg acc aag aac ctg gag aag tac atc cag atc 672Ala Glu Phe Glu Ile Leu Thr Lys Asn Leu Glu Lys Tyr Ile Gln Ile 210 215 220 gac gag aag ctg gac gag ttc gtg gag cac gcc gag aac aac aag cat 720Asp Glu Lys Leu Asp Glu Phe Val Glu His Ala Glu Asn Asn Lys His 225 230 235 240 atc gcc tct atc gcc ctg aac aac ctg aat aag agc ggc ctg gtg gga 768Ile Ala Ser Ile Ala Leu Asn Asn Leu Asn Lys Ser Gly Leu Val Gly 245 250 255 gag ggc gag agc aaa aag atc ctg gct aag atg ctg aac atg gac ggc 816Glu Gly Glu Ser Lys Lys Ile Leu Ala Lys Met Leu Asn Met Asp Gly 260 265 270 atg gat ctg ctg ggc gtg gac ccc aag cac gtg tgc gtg gac acc aga 864Met Asp Leu Leu Gly Val Asp Pro Lys His Val Cys Val Asp Thr Arg 275 280 285 gac atc ccc aag aac gcc ggc tgc ttc agg gac gac aac ggc acc gag 912Asp Ile Pro Lys Asn Ala Gly Cys Phe Arg Asp Asp Asn Gly Thr Glu 290 295 300 gag tgg aga tgt ctg ctg ggc tac aag aag ggc gag ggc aac acc tgc 960Glu Trp Arg Cys Leu Leu Gly Tyr Lys Lys Gly Glu Gly Asn Thr Cys 305 310 315 320 gtg gag aac aat aac ccc acc tgc gac atc aac aac ggc ggc tgc gac 1008Val Glu Asn Asn Asn Pro Thr Cys Asp Ile Asn Asn Gly Gly Cys Asp 325 330 335 ccc acc gcc agc tgc cag aac gcc gag agc acc gag aac tcc aag aag 1056Pro Thr Ala Ser Cys Gln Asn Ala Glu Ser Thr Glu Asn Ser Lys Lys 340 345 350 atc atc tgc acc tgc aag gag ccc acc ccc aac gcc tac tac gag ggc 1104Ile Ile Cys Thr Cys Lys Glu Pro Thr Pro Asn Ala Tyr Tyr Glu Gly 355 360 365 gtg ttc tgc agc agc agc tcc ttc atg ggc tga tga 1140Val Phe Cys Ser Ser Ser Ser Phe Met Gly 370 375 36378PRTArtificialSynthetic Construct 36Met Leu Leu Ser Val Pro Leu Leu Leu Gly Leu Leu Gly Leu Ala Val 1 5 10 15 Ala Ala Pro Glu Lys Asp Ile Leu Ser Glu Phe Thr Asn Glu Ser Leu 20 25 30 Tyr Val Tyr Thr Lys Arg Leu Gly Ser Thr Tyr Lys Ser Leu Lys Lys 35 40 45 His Met Leu Arg Glu Phe Ser Thr Ile Lys Glu Asp Met Thr Asn Gly 50 55 60 Leu Asn Asn Lys Ser Gln Lys Arg Asn Asp Phe Leu Glu Val Leu Ser 65 70 75 80 His Glu Leu Asp Leu Phe Lys Asp Leu Ser Thr Asn Lys Tyr Val Ile 85 90 95 Arg Asn Pro Tyr Gln Leu Leu Asp Asn Asp Lys Lys Asp Lys Gln Ile 100 105 110 Val Asn Leu Lys Tyr Ala Thr Lys Gly Ile Asn Glu Asp Ile Glu Thr 115 120 125 Thr Thr Asp Gly Ile Lys Phe Phe Asn Lys Met Val Glu Leu Tyr Asn 130 135 140 Thr Gln Leu Ala Ala Val Lys Glu Gln Ile Ala Thr Ile Glu Ala Glu 145 150 155 160 Thr Asn Asp Thr Asn Lys Glu Glu Lys Lys Lys Tyr Ile Pro Ile Leu 165 170 175 Glu Asp Leu Lys Gly Leu Tyr Glu Thr Val Ile Gly Gln Ala Glu Glu 180 185 190 Tyr Ser Glu Glu Leu Gln Asn Arg Leu Asp Asn Tyr Lys Asn Glu Lys 195 200 205 Ala Glu Phe Glu Ile Leu Thr Lys Asn Leu Glu Lys Tyr Ile Gln Ile 210 215 220 Asp Glu Lys Leu Asp Glu Phe Val Glu His Ala Glu Asn Asn Lys His 225 230 235 240 Ile Ala Ser Ile Ala Leu Asn Asn Leu Asn Lys Ser Gly Leu Val Gly 245 250 255 Glu Gly Glu Ser Lys Lys Ile Leu Ala Lys Met Leu Asn Met Asp Gly 260 265 270 Met Asp Leu Leu Gly Val Asp Pro Lys His Val Cys Val Asp Thr Arg 275 280 285 Asp Ile Pro Lys Asn Ala Gly Cys Phe Arg Asp Asp Asn Gly Thr Glu 290 295 300 Glu Trp Arg Cys Leu Leu Gly Tyr Lys Lys Gly Glu Gly Asn Thr Cys 305 310 315 320 Val Glu Asn Asn Asn Pro Thr Cys Asp Ile Asn Asn Gly Gly Cys Asp 325 330 335 Pro Thr Ala Ser Cys Gln Asn Ala Glu Ser Thr Glu Asn Ser Lys Lys 340 345 350 Ile Ile Cys Thr Cys Lys Glu Pro Thr Pro Asn Ala Tyr Tyr Glu Gly 355 360 365 Val Phe Cys Ser Ser Ser Ser Phe Met Gly 370 375 374300DNAArtificialplasmid pTRIP-deltaU3-CMV-Hep17 CO-WPRE 37tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca 60cacaaggcta cttccctgat tagcagaact acacaccagg gccagggatc agatatccac 120tgacctttgg atggtgctac aagctagtac cagttgagcc agagaagtta gaagaagcca 180acaaaggaga gaacaccagc ttgttacaac ctgtgagcct gcatgggatg gatgacccgg 240agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac ggtggcccga 300gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa gggactttcc 360gctgggggac tttccaggga ggcgtggcct gggcgggact ggggagtggc gagccctcag 420atcctgcata taagcagctg ctttttgcct gtactgggtc tctctggtta gaccagatct 480gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 540cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 600tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa 660agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac 720ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 780gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg 840ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg 900ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg 960ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag 1020atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga 1080caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca 1140gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg 1200aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa 1260agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt 1320tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca 1380gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc 1440aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg 1500ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac 1560tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga 1620tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa 1680tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg 1740aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata 1800taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac 1860tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc 1920caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca 1980gagacagatc cattcgatta gtgaacggat ctcgacggta tcgccgaatt cacaaatggc 2040agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag 2100aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa 2160aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc tgatacgcgt 2220ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 2280ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 2340ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 2400tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 2460tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 2520cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 2580ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 2640aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 2700taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc 2760ctggagacgc catccacgct gttttgacct ccatagaaga caccgcgatc ggatccgcca 2820ccatgaagat caatatcgcc agcatcatct ttatcatctt cagcctgtgc ctggtcaacg 2880acgcctacgg caagaacaag tacgggaaga acggcaagta cggcagccag aacgtgatca 2940agaaacacgg cgagcccgtg atcaacgtgc aggacctgat cagcgacatg gtccggaaag 3000aggaagagat cgtcaagctg accaagaaca agaagagcct gaggaagatc aacgtggccc 3060tggccaccgc cctgagcgtg gtgtccgcca tcctgctggg cggagccggc ctggtcatgt 3120acaacaccga gaagggcaga aggcccttcc agatcggcaa gagcaagaaa ggcggcagcg 3180ccatggccag ggacagcagc ttccccatga acgaggaaag ccccctgggc ttcagccccg 3240aggaaatgga agccgtggcc agcaagttcc gggagagcat gctgaaggac ggcgtgcctg 3300cccccagcaa cacccccaac gtgcagaact gatgactcga gctcaagctt cgaattcccg 3360ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg 3420ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc 3480gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt 3540tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca 3600ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc 3660ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc 3720tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc 3780tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc 3840tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc 3900ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg cgtcgacgcg 3960tgaattcggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt 4020taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatcgtcgag 4080agatgctgca tataagcagc tgctttttgc ttgtactggg tctctctggt tagaccagat 4140ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt 4200gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc 4260cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 430038513DNAArtificialSequence for transgene for Hep17-CO 38atg aag atc aat atc gcc agc atc atc ttt atc atc ttc agc ctg tgc 48Met Lys Ile Asn Ile Ala Ser Ile Ile Phe Ile Ile Phe Ser Leu Cys 1 5 10 15 ctg gtc aac gac gcc tac ggc aag aac aag tac ggg aag aac ggc aag 96Leu Val Asn Asp Ala Tyr Gly Lys Asn Lys Tyr Gly Lys Asn Gly Lys 20 25 30 tac ggc agc cag aac gtg atc aag aaa cac ggc gag ccc gtg atc aac 144Tyr Gly Ser Gln Asn Val Ile Lys Lys His Gly Glu Pro Val Ile Asn 35 40 45 gtg cag gac ctg atc agc gac atg gtc cgg aaa gag gaa gag atc gtc 192Val Gln Asp Leu Ile Ser Asp Met Val Arg Lys Glu Glu Glu Ile Val 50 55 60 aag ctg acc aag aac aag aag agc ctg agg aag atc aac gtg gcc ctg 240Lys Leu Thr Lys Asn Lys Lys Ser Leu Arg Lys Ile Asn Val Ala Leu 65 70 75 80 gcc acc gcc ctg agc gtg gtg tcc gcc atc ctg ctg ggc gga gcc ggc 288Ala Thr Ala Leu Ser Val Val Ser Ala Ile Leu Leu Gly Gly Ala Gly 85 90 95 ctg gtc atg tac aac acc gag aag ggc aga agg ccc ttc cag atc ggc 336Leu Val Met Tyr Asn Thr Glu Lys Gly Arg Arg Pro Phe Gln Ile Gly 100 105 110 aag agc aag aaa ggc ggc agc gcc atg gcc agg gac agc agc ttc ccc 384Lys Ser Lys Lys Gly Gly Ser Ala Met Ala Arg Asp Ser Ser Phe Pro 115 120 125 atg aac gag gaa agc ccc ctg ggc ttc agc ccc gag gaa atg gaa gcc 432Met Asn Glu Glu Ser Pro Leu Gly Phe Ser Pro Glu Glu Met Glu Ala 130 135 140 gtg gcc agc aag ttc cgg gag agc atg ctg aag gac ggc gtg cct gcc 480Val Ala Ser Lys Phe Arg Glu Ser Met Leu Lys Asp Gly Val Pro Ala 145 150 155 160 ccc agc aac acc ccc aac gtg cag aac tga tga 513Pro Ser Asn Thr Pro Asn Val Gln Asn 165 39169PRTArtificialSynthetic Construct 39Met Lys Ile Asn Ile Ala Ser Ile Ile Phe Ile Ile Phe Ser Leu Cys 1 5 10 15 Leu Val Asn Asp Ala Tyr Gly Lys Asn Lys Tyr Gly Lys Asn Gly Lys 20 25 30 Tyr Gly Ser Gln Asn Val Ile Lys Lys His Gly Glu Pro Val Ile Asn 35 40 45 Val Gln Asp Leu Ile Ser Asp Met Val Arg Lys Glu Glu Glu Ile Val 50 55 60 Lys Leu Thr Lys Asn Lys Lys Ser Leu Arg Lys Ile Asn Val Ala Leu 65 70 75 80 Ala Thr Ala Leu Ser Val Val Ser Ala Ile Leu Leu Gly Gly Ala Gly 85 90 95 Leu Val Met Tyr Asn Thr Glu Lys Gly Arg Arg Pro Phe Gln Ile Gly 100 105 110 Lys Ser Lys Lys Gly Gly Ser Ala Met Ala Arg Asp Ser Ser Phe Pro 115 120 125 Met Asn Glu Glu Ser Pro Leu Gly Phe Ser Pro Glu Glu Met Glu Ala 130 135 140 Val Ala Ser Lys Phe Arg Glu Ser Met Leu Lys Asp Gly Val Pro Ala 145 150 155 160 Pro Ser Asn Thr Pro Asn Val Gln Asn 165 404261DNAArtificialplasmid pTRIP-deltaU3-CMV-Hep17 deltaSP CO-WPRE 40tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca 60cacaaggcta cttccctgat tagcagaact acacaccagg gccagggatc agatatccac 120tgacctttgg atggtgctac aagctagtac cagttgagcc agagaagtta gaagaagcca 180acaaaggaga gaacaccagc ttgttacaac ctgtgagcct gcatgggatg gatgacccgg 240agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac ggtggcccga 300gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa gggactttcc 360gctgggggac tttccaggga ggcgtggcct gggcgggact ggggagtggc gagccctcag 420atcctgcata taagcagctg ctttttgcct gtactgggtc tctctggtta gaccagatct 480gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 540cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 600tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa 660agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac 720ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 780gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg 840ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg 900ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg 960ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag 1020atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga 1080caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca 1140gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg 1200aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa 1260agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt 1320tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca 1380gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc 1440aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg 1500ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac 1560tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga 1620tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa 1680tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg 1740aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata 1800taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac 1860tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc 1920caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca 1980gagacagatc cattcgatta gtgaacggat ctcgacggta tcgccgaatt cacaaatggc 2040agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag 2100aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa 2160aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc tgatacgcgt 2220ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 2280ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 2340ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 2400tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 2460tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 2520cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 2580ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 2640aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 2700taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc 2760ctggagacgc catccacgct

gttttgacct ccatagaaga caccgcgatc ggatccgcca 2820ccatgctgtg cctggtcaac gacgcctacg gcaagaacaa gtacgggaag aacggcaagt 2880acggcagcca gaacgtgatc aagaaacacg gcgagcccgt gatcaacgtg caggacctga 2940tcagcgacat ggtccggaaa gaggaagaga tcgtcaagct gaccaagaac aagaagagcc 3000tgaggaagat caacgtggcc ctggccaccg ccctgagcgt ggtgtccgcc atcctgctgg 3060gcggagccgg cctggtcatg tacaacaccg agaagggcag aaggcccttc cagatcggca 3120agagcaagaa aggcggcagc gccatggcca gggacagcag cttccccatg aacgaggaaa 3180gccccctggg cttcagcccc gaggaaatgg aagccgtggc cagcaagttc cgggagagca 3240tgctgaagga cggcgtgcct gcccccagca acacccccaa cgtgcagaac tgatgactcg 3300agctcaagct tcgaattccc gataatcaac ctctggatta caaaatttgt gaaagattga 3360ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt 3420tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt 3480tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg 3540tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg 3600ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc 3660gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaagc 3720tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct 3780tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg 3840ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg 3900ccgcctcccc gcgtcgacgc gtgaattcgg tacctttaag accaatgact tacaaggcag 3960ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc 4020aacgaagaca agatcgtcga gagatgctgc atataagcag ctgctttttg cttgtactgg 4080gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact 4140gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg 4200tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag 4260t 426141474DNAArtificialSequence of transgene from Hep17deltaSP CO 41atg ctg tgc ctg gtc aac gac gcc tac ggc aag aac aag tac ggg aag 48Met Leu Cys Leu Val Asn Asp Ala Tyr Gly Lys Asn Lys Tyr Gly Lys 1 5 10 15 aac ggc aag tac ggc agc cag aac gtg atc aag aaa cac ggc gag ccc 96Asn Gly Lys Tyr Gly Ser Gln Asn Val Ile Lys Lys His Gly Glu Pro 20 25 30 gtg atc aac gtg cag gac ctg atc agc gac atg gtc cgg aaa gag gaa 144Val Ile Asn Val Gln Asp Leu Ile Ser Asp Met Val Arg Lys Glu Glu 35 40 45 gag atc gtc aag ctg acc aag aac aag aag agc ctg agg aag atc aac 192Glu Ile Val Lys Leu Thr Lys Asn Lys Lys Ser Leu Arg Lys Ile Asn 50 55 60 gtg gcc ctg gcc acc gcc ctg agc gtg gtg tcc gcc atc ctg ctg ggc 240Val Ala Leu Ala Thr Ala Leu Ser Val Val Ser Ala Ile Leu Leu Gly 65 70 75 80 gga gcc ggc ctg gtc atg tac aac acc gag aag ggc aga agg ccc ttc 288Gly Ala Gly Leu Val Met Tyr Asn Thr Glu Lys Gly Arg Arg Pro Phe 85 90 95 cag atc ggc aag agc aag aaa ggc ggc agc gcc atg gcc agg gac agc 336Gln Ile Gly Lys Ser Lys Lys Gly Gly Ser Ala Met Ala Arg Asp Ser 100 105 110 agc ttc ccc atg aac gag gaa agc ccc ctg ggc ttc agc ccc gag gaa 384Ser Phe Pro Met Asn Glu Glu Ser Pro Leu Gly Phe Ser Pro Glu Glu 115 120 125 atg gaa gcc gtg gcc agc aag ttc cgg gag agc atg ctg aag gac ggc 432Met Glu Ala Val Ala Ser Lys Phe Arg Glu Ser Met Leu Lys Asp Gly 130 135 140 gtg cct gcc ccc agc aac acc ccc aac gtg cag aac tga tga 474Val Pro Ala Pro Ser Asn Thr Pro Asn Val Gln Asn 145 150 155 42156PRTArtificialSynthetic Construct 42Met Leu Cys Leu Val Asn Asp Ala Tyr Gly Lys Asn Lys Tyr Gly Lys 1 5 10 15 Asn Gly Lys Tyr Gly Ser Gln Asn Val Ile Lys Lys His Gly Glu Pro 20 25 30 Val Ile Asn Val Gln Asp Leu Ile Ser Asp Met Val Arg Lys Glu Glu 35 40 45 Glu Ile Val Lys Leu Thr Lys Asn Lys Lys Ser Leu Arg Lys Ile Asn 50 55 60 Val Ala Leu Ala Thr Ala Leu Ser Val Val Ser Ala Ile Leu Leu Gly 65 70 75 80 Gly Ala Gly Leu Val Met Tyr Asn Thr Glu Lys Gly Arg Arg Pro Phe 85 90 95 Gln Ile Gly Lys Ser Lys Lys Gly Gly Ser Ala Met Ala Arg Asp Ser 100 105 110 Ser Phe Pro Met Asn Glu Glu Ser Pro Leu Gly Phe Ser Pro Glu Glu 115 120 125 Met Glu Ala Val Ala Ser Lys Phe Arg Glu Ser Met Leu Lys Asp Gly 130 135 140 Val Pro Ala Pro Ser Asn Thr Pro Asn Val Gln Asn 145 150 155 434897DNAArtificialPlasmid pTRIP-DeltaU3-CMV-CSP CO WPRE 43tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca 60cacaaggcta cttccctgat tagcagaact acacaccagg gccagggatc agatatccac 120tgacctttgg atggtgctac aagctagtac cagttgagcc agagaagtta gaagaagcca 180acaaaggaga gaacaccagc ttgttacaac ctgtgagcct gcatgggatg gatgacccgg 240agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac ggtggcccga 300gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa gggactttcc 360gctgggggac tttccaggga ggcgtggcct gggcgggact ggggagtggc gagccctcag 420atcctgcata taagcagctg ctttttgcct gtactgggtc tctctggtta gaccagatct 480gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 540cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 600tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa 660agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac 720ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 780gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg 840ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg 900ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg 960ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag 1020atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga 1080caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca 1140gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg 1200aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa 1260agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt 1320tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca 1380gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc 1440aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg 1500ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac 1560tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga 1620tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa 1680tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg 1740aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata 1800taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac 1860tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc 1920caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca 1980gagacagatc cattcgatta gtgaacggat ctcgacggta tcgccgaatt cacaaatggc 2040agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag 2100aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa 2160aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc tgatacgcgt 2220ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 2280ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 2340ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 2400tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 2460tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 2520cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 2580ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 2640aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 2700taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc 2760ctggagacgc catccacgct gttttgacct ccatagaaga caccgcgatc ggatccgcca 2820ccatgaagaa atgcaccatc ctggtggtgg ccagcctgct gctggtcgat agcctgctgc 2880ccggctacgg ccagaataag agcgtgcagg cccagcggaa cctgaacgag ctgtgctaca 2940acgaggaaaa cgacaacaag ctgtaccacg tgctgaacag caagaacggc aagatctaca 3000accggaacat cgtgaacagg ctgctgggcg acgctctgaa cggcaagccc gaggaaaaga 3060aggacgaccc ccccaaggac ggcaacaagg acgacctgcc caaagaagag aagaaagacg 3120atctgcctaa agaggaaaaa aaagacgatc ctcctaagga ccccaagaag gatgaccctc 3180ctaaagaggc ccagaacaag ctgaaccagc ccgtggtggc cgacgagaac gtggatcagg 3240gacctggcgc ccctcagggc ccaggcgctc cacagggacc cggggcaccc caggggcctg 3300gggccccaca gggaccaggg gctcctcagg gccctggcgc acctcagggg ccaggggccc 3360ctcaggggcc tggcgctccc cagggacctg gcgcaccaca gggccctggg gctccccagg 3420gcccaggcgc ccctcaggga ccaggcgcac cccagggacc cggcgctcct cagggacctg 3480gggctccaca ggggccaggc gcaccacagg aacctcccca gcagcctcct cagcagccac 3540cccagcagcc ccctcagcag cctcctcagc agcccccaca gcagcctcca cagcagccta 3600gaccccagcc cgacggcaat aacaacaaca ataataacaa cggcaacaac aacgaggaca 3660gctacgtgcc cagcgccgag cagatcctgg aattcgtgaa gcagatcagc agccagctga 3720ccgaagagtg gagccagtgc agcgtgacat gcggctctgg cgtgagagtg cggaagcgga 3780agaacgtgaa caagcagccc gagaacctga ccctggaaga tatcgacacc gagatctgca 3840agatggacaa gtgcagcagc atcttcaaca tcgtgtccaa cagcctgggc ttcgtgatcc 3900tgctggtgct ggtgttcttc aactgatgac tcgagctcaa gcttcgaatt cccgataatc 3960aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt 4020ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg 4080ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc 4140ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt 4200ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg 4260ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg 4320gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct 4380gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc 4440cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc 4500ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga cgcgtgaatt 4560cggtaccttt aagaccaatg acttacaagg cagctgagat cttagccact ttttaaaaga 4620aaagggggga ctggaagggc taattcactc ccaacgaaga caagatcgtc gagagatgct 4680gcatataagc agctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc 4740tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga 4800gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga 4860cccttttagt cagtgtggaa aatctctagc agtattt 4897441107DNAArtificialSequence of transgene for CSP-CO 44atgaagaaat gcaccatcct ggtggtggcc agcctgctgc tggtcgatag cctgctgccc 60ggctacggcc agaataagag cgtgcaggcc cagcggaacc tgaacgagct gtgctacaac 120gaggaaaacg acaacaagct gtaccacgtg ctgaacagca agaacggcaa gatctacaac 180cggaacatcg tgaacaggct gctgggcgac gctctgaacg gcaagcccga ggaaaagaag 240gacgaccccc ccaaggacgg caacaaggac gacctgccca aagaagagaa gaaagacgat 300ctgcctaaag aggaaaaaaa agacgatcct cctaaggacc ccaagaagga tgaccctcct 360aaagaggccc agaacaagct gaaccagccc gtggtggccg acgagaacgt ggatcaggga 420cctggcgccc ctcagggccc aggcgctcca cagggacccg gggcacccca ggggcctggg 480gccccacagg gaccaggggc tcctcagggc cctggcgcac ctcaggggcc aggggcccct 540caggggcctg gcgctcccca gggacctggc gcaccacagg gccctggggc tccccagggc 600ccaggcgccc ctcagggacc aggcgcaccc cagggacccg gcgctcctca gggacctggg 660gctccacagg ggccaggcgc accacaggaa cctccccagc agcctcctca gcagccaccc 720cagcagcccc ctcagcagcc tcctcagcag cccccacagc agcctccaca gcagcctaga 780ccccagcccg acggcaataa caacaacaat aataacaacg gcaacaacaa cgaggacagc 840tacgtgccca gcgccgagca gatcctggaa ttcgtgaagc agatcagcag ccagctgacc 900gaagagtgga gccagtgcag cgtgacatgc ggctctggcg tgagagtgcg gaagcggaag 960aacgtgaaca agcagcccga gaacctgacc ctggaagata tcgacaccga gatctgcaag 1020atggacaagt gcagcagcat cttcaacatc gtgtccaaca gcctgggctt cgtgatcctg 1080ctggtgctgg tgttcttcaa ctgatga 1107454840DNAArtificialplasmid pTRIP-deltaU3-CMV-CSP deltaSP CO-WPRE 45tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca 60cacaaggcta cttccctgat tagcagaact acacaccagg gccagggatc agatatccac 120tgacctttgg atggtgctac aagctagtac cagttgagcc agagaagtta gaagaagcca 180acaaaggaga gaacaccagc ttgttacaac ctgtgagcct gcatgggatg gatgacccgg 240agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac ggtggcccga 300gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa gggactttcc 360gctgggggac tttccaggga ggcgtggcct gggcgggact ggggagtggc gagccctcag 420atcctgcata taagcagctg ctttttgcct gtactgggtc tctctggtta gaccagatct 480gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 540cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 600tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa 660agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac 720ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 780gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg 840ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg 900ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg 960ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag 1020atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga 1080caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca 1140gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg 1200aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa 1260agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt 1320tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca 1380gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc 1440aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg 1500ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac 1560tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga 1620tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa 1680tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg 1740aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata 1800taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac 1860tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc 1920caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca 1980gagacagatc cattcgatta gtgaacggat ctcgacggta tcgccgaatt cacaaatggc 2040agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag 2100aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa 2160aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc tgatacgcgt 2220ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 2280ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 2340ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 2400tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 2460tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 2520cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 2580ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 2640aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 2700taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc 2760ctggagacgc catccacgct gttttgacct ccatagaaga caccgcgatc ggatccgcca 2820ccatgcccgg ctacggccag aataagagcg tgcaggccca gcggaacctg aacgagctgt 2880gctacaacga ggaaaacgac aacaagctgt accacgtgct gaacagcaag aacggcaaga 2940tctacaaccg gaacatcgtg aacaggctgc tgggcgacgc tctgaacggc aagcccgagg 3000aaaagaagga cgaccccccc aaggacggca acaaggacga cctgcccaaa gaagagaaga 3060aagacgatct gcctaaagag gaaaaaaaag acgatcctcc taaggacccc aagaaggatg 3120accctcctaa agaggcccag aacaagctga accagcccgt ggtggccgac gagaacgtgg 3180atcagggacc tggcgcccct cagggcccag gcgctccaca gggacccggg gcaccccagg 3240ggcctggggc cccacaggga ccaggggctc ctcagggccc tggcgcacct caggggccag 3300gggcccctca ggggcctggc gctccccagg gacctggcgc accacagggc cctggggctc 3360cccagggccc aggcgcccct cagggaccag gcgcacccca gggacccggc gctcctcagg 3420gacctggggc tccacagggg ccaggcgcac cacaggaacc tccccagcag cctcctcagc 3480agccacccca gcagccccct cagcagcctc ctcagcagcc cccacagcag cctccacagc 3540agcctagacc ccagcccgac ggcaataaca acaacaataa taacaacggc aacaacaacg 3600aggacagcta cgtgcccagc gccgagcaga tcctggaatt cgtgaagcag atcagcagcc 3660agctgaccga agagtggagc cagtgcagcg tgacatgcgg ctctggcgtg agagtgcgga 3720agcggaagaa cgtgaacaag cagcccgaga acctgaccct ggaagatatc gacaccgaga 3780tctgcaagat ggacaagtgc agcagcatct tcaacatcgt gtccaacagc ctgggcttcg 3840tgatcctgct ggtgctggtg ttcttcaact gatgactcga gctcaagctt cgaattcccg 3900ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg 3960ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc 4020gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt 4080tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca 4140ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc 4200ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc 4260tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc 4320tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc

4380tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc 4440ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg cgtcgacgcg 4500tgaattcggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt 4560taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatcgtcgag 4620agatgctgca tataagcagc tgctttttgc ttgtactggg tctctctggt tagaccagat 4680ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt 4740gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc 4800cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 4840461053DNAArtificialSequence of transgene for CSP-deltaSP 46atgcccggct acggccagaa taagagcgtg caggcccagc ggaacctgaa cgagctgtgc 60tacaacgagg aaaacgacaa caagctgtac cacgtgctga acagcaagaa cggcaagatc 120tacaaccgga acatcgtgaa caggctgctg ggcgacgctc tgaacggcaa gcccgaggaa 180aagaaggacg acccccccaa ggacggcaac aaggacgacc tgcccaaaga agagaagaaa 240gacgatctgc ctaaagagga aaaaaaagac gatcctccta aggaccccaa gaaggatgac 300cctcctaaag aggcccagaa caagctgaac cagcccgtgg tggccgacga gaacgtggat 360cagggacctg gcgcccctca gggcccaggc gctccacagg gacccggggc accccagggg 420cctggggccc cacagggacc aggggctcct cagggccctg gcgcacctca ggggccaggg 480gcccctcagg ggcctggcgc tccccaggga cctggcgcac cacagggccc tggggctccc 540cagggcccag gcgcccctca gggaccaggc gcaccccagg gacccggcgc tcctcaggga 600cctggggctc cacaggggcc aggcgcacca caggaacctc cccagcagcc tcctcagcag 660ccaccccagc agccccctca gcagcctcct cagcagcccc cacagcagcc tccacagcag 720cctagacccc agcccgacgg caataacaac aacaataata acaacggcaa caacaacgag 780gacagctacg tgcccagcgc cgagcagatc ctggaattcg tgaagcagat cagcagccag 840ctgaccgaag agtggagcca gtgcagcgtg acatgcggct ctggcgtgag agtgcggaag 900cggaagaacg tgaacaagca gcccgagaac ctgaccctgg aagatatcga caccgagatc 960tgcaagatgg acaagtgcag cagcatcttc aacatcgtgt ccaacagcct gggcttcgtg 1020atcctgctgg tgctggtgtt cttcaactga tga 1053474858DNAArtificialPlasmid pTRIP-deltaU3-CMV-CSP deltaGPI CO-WPRE 47tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca 60cacaaggcta cttccctgat tagcagaact acacaccagg gccagggatc agatatccac 120tgacctttgg atggtgctac aagctagtac cagttgagcc agagaagtta gaagaagcca 180acaaaggaga gaacaccagc ttgttacaac ctgtgagcct gcatgggatg gatgacccgg 240agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac ggtggcccga 300gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa gggactttcc 360gctgggggac tttccaggga ggcgtggcct gggcgggact ggggagtggc gagccctcag 420atcctgcata taagcagctg ctttttgcct gtactgggtc tctctggtta gaccagatct 480gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 540cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 600tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa 660agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac 720ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 780gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg 840ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg 900ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg 960ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag 1020atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga 1080caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca 1140gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg 1200aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa 1260agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt 1320tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca 1380gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc 1440aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg 1500ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac 1560tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga 1620tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa 1680tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg 1740aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata 1800taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac 1860tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc 1920caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca 1980gagacagatc cattcgatta gtgaacggat ctcgacggta tcgccgaatt cacaaatggc 2040agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag 2100aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa 2160aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc tgatacgcgt 2220ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 2280ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 2340ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 2400tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 2460tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 2520cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 2580ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 2640aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 2700taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc 2760ctggagacgc catccacgct gttttgacct ccatagaaga caccgcgatc ggatccgcca 2820ccatgaagaa atgcaccatc ctggtggtgg ccagcctgct gctggtcgat agcctgctgc 2880ccggctacgg ccagaataag agcgtgcagg cccagcggaa cctgaacgag ctgtgctaca 2940acgaggaaaa cgacaacaag ctgtaccacg tgctgaacag caagaacggc aagatctaca 3000accggaacat cgtgaacagg ctgctgggcg acgctctgaa cggcaagccc gaggaaaaga 3060aggacgaccc ccccaaggac ggcaacaagg acgacctgcc caaagaagag aagaaagacg 3120atctgcctaa agaggaaaaa aaagacgatc ctcctaagga ccccaagaag gatgaccctc 3180ctaaagaggc ccagaacaag ctgaaccagc ccgtggtggc cgacgagaac gtggatcagg 3240gacctggcgc ccctcagggc ccaggcgctc cacagggacc cggggcaccc caggggcctg 3300gggccccaca gggaccaggg gctcctcagg gccctggcgc acctcagggg ccaggggccc 3360ctcaggggcc tggcgctccc cagggacctg gcgcaccaca gggccctggg gctccccagg 3420gcccaggcgc ccctcaggga ccaggcgcac cccagggacc cggcgctcct cagggacctg 3480gggctccaca ggggccaggc gcaccacagg aacctcccca gcagcctcct cagcagccac 3540cccagcagcc ccctcagcag cctcctcagc agcccccaca gcagcctcca cagcagccta 3600gaccccagcc cgacggcaat aacaacaaca ataataacaa cggcaacaac aacgaggaca 3660gctacgtgcc cagcgccgag cagatcctgg aattcgtgaa gcagatcagc agccagctga 3720ccgaagagtg gagccagtgc agcgtgacat gcggctctgg cgtgagagtg cggaagcgga 3780agaacgtgaa caagcagccc gagaacctga ccctggaaga tatcgacacc gagatctgca 3840agatggacaa gtgcagcagc atcttcaaca tcgtgtccaa cagcctgtga tgactcgagc 3900tcaagcttcg aattcccgat aatcaacctc tggattacaa aatttgtgaa agattgactg 3960gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt 4020atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc 4080tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt 4140ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga 4200ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct 4260gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaagctga 4320cgtcctttcc atggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct 4380gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc 4440tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg 4500cctccccgcg tcgacgcgtg aattcggtac ctttaagacc aatgacttac aaggcagctg 4560tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac 4620gaagacaaga tcgtcgagag atgctgcata taagcagctg ctttttgctt gtactgggtc 4680tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct 4740taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga 4800ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagt 4858481071DNAArtificialSequence of transgene for CSP-delta GPI CO 48atgaagaaat gcaccatcct ggtggtggcc agcctgctgc tggtcgatag cctgctgccc 60ggctacggcc agaataagag cgtgcaggcc cagcggaacc tgaacgagct gtgctacaac 120gaggaaaacg acaacaagct gtaccacgtg ctgaacagca agaacggcaa gatctacaac 180cggaacatcg tgaacaggct gctgggcgac gctctgaacg gcaagcccga ggaaaagaag 240gacgaccccc ccaaggacgg caacaaggac gacctgccca aagaagagaa gaaagacgat 300ctgcctaaag aggaaaaaaa agacgatcct cctaaggacc ccaagaagga tgaccctcct 360aaagaggccc agaacaagct gaaccagccc gtggtggccg acgagaacgt ggatcaggga 420cctggcgccc ctcagggccc aggcgctcca cagggacccg gggcacccca ggggcctggg 480gccccacagg gaccaggggc tcctcagggc cctggcgcac ctcaggggcc aggggcccct 540caggggcctg gcgctcccca gggacctggc gcaccacagg gccctggggc tccccagggc 600ccaggcgccc ctcagggacc aggcgcaccc cagggacccg gcgctcctca gggacctggg 660gctccacagg ggccaggcgc accacaggaa cctccccagc agcctcctca gcagccaccc 720cagcagcccc ctcagcagcc tcctcagcag cccccacagc agcctccaca gcagcctaga 780ccccagcccg acggcaataa caacaacaat aataacaacg gcaacaacaa cgaggacagc 840tacgtgccca gcgccgagca gatcctggaa ttcgtgaagc agatcagcag ccagctgacc 900gaagagtgga gccagtgcag cgtgacatgc ggctctggcg tgagagtgcg gaagcggaag 960aacgtgaaca agcagcccga gaacctgacc ctggaagata tcgacaccga gatctgcaag 1020atggacaagt gcagcagcat cttcaacatc gtgtccaaca gcctgtgatg a 107149442PRTPlasmodium falciparumMISC_FEATURE(1)..(442)CSP protein GenBank M15505.1 49Met Met Arg Lys Leu Ala Ile Leu Ser Val Ser Ser Phe Leu Phe Val 1 5 10 15 Glu Ala Leu Phe Gln Glu Tyr Gln Cys Tyr Gly Ser Ser Ser Asn Thr 20 25 30 Arg Val Leu Asn Glu Leu Asn Tyr Asp Asn Ala Gly Thr Asn Leu Tyr 35 40 45 Asn Glu Leu Glu Met Asn Tyr Tyr Gly Lys Gln Glu Asn Trp Tyr Ser 50 55 60 Leu Lys Lys Asn Ser Arg Ser Leu Gly Glu Asn Asp Asp Gly Asp Asn 65 70 75 80 Asp Asn Gly Asn Asn Asn Asn Gly Asn Asn Asn Asn Gly Asp Asn Gly 85 90 95 Arg Glu Gly Lys Asp Glu Asp Lys Arg Asp Gly Asn Asn Glu Asp Asn 100 105 110 Glu Lys Leu Arg Lys Pro Lys His Lys Lys Leu Lys Gln Pro Gly Asp 115 120 125 Gly Asn Pro Asp Pro Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn 130 135 140 Pro Asn Val Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 145 150 155 160 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 165 170 175 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 180 185 190 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 195 200 205 Pro Asn Val Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 210 215 220 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 225 230 235 240 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 245 250 255 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 260 265 270 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 275 280 285 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn 290 295 300 Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Lys Asn 305 310 315 320 Asn Gln Gly Asn Gly Gln Gly His Asn Met Pro Asn Asp Pro Asn Arg 325 330 335 Asn Val Asp Glu Asn Ala Asn Ala Asn Asn Ala Val Lys Asn Asn Asn 340 345 350 Asn Glu Glu Pro Ser Asp Lys His Ile Glu Gln Tyr Leu Lys Lys Ile 355 360 365 Gln Asn Ser Leu Ser Thr Glu Trp Ser Pro Cys Ser Val Thr Cys Gly 370 375 380 Asn Gly Ile Gln Val Arg Ile Lys Pro Gly Ser Ala Asp Lys Pro Lys 385 390 395 400 Asp Gln Leu Asp Tyr Glu Asn Asp Ile Glu Lys Lys Ile Cys Lys Met 405 410 415 Glu Lys Cys Ser Ser Val Phe Asn Val Val Asn Ser Ser Ile Gly Leu 420 425 430 Ile Met Val Leu Ser Phe Leu Phe Leu Asn 435 440 501669DNAPlasmodium falciparummisc_feature(1)..(1669)CSP gene GenBank M15505.1 50gtagaaacca cgtaatatta taaattacaa ttcatgatga gaaaattagc tattttatct 60gtttcttcct ttttatttgt tgaggcctta ttccaggaat accagtgcta tggaagttcg 120tcaaacacaa gggttctaaa tgaattaaat tatgataatg caggcactaa tttatataat 180gaattagaaa tgaattatta tgggaaacag gaaaattggt atagtcttaa aaaaaatagt 240agatcacttg gagaaaatga tgatggagat aatgataatg gaaataataa taatggaaat 300aataataatg gagataatgg tcgtgaaggt aaagatgaag ataaaagaga tggaaataac 360gaagacaacg agaaattaag gaaaccaaaa cataaaaaat taaagcaacc aggggatggt 420aatcctgatc caaatgccaa cccaaatgta gatccaaatg ccaacccaaa tgtagatcca 480aatgcaaacc caaatgcaaa cccaaatgca aacccaaatg caaacccaaa tgcaaaccca 540aatgcaaacc caaatgcaaa cccaaatgca aacccaaatg caaacccaaa tgcaaaccca 600aatgcaaacc caaatgcaaa cccaaatgca aacccaaatg caaacccaaa tgcaaaccca 660aacgtagatc ctaatgcaaa tccaaatgca aacccaaatg caaacccaaa cgcaaaccca 720aatgcaaatc ctaatgcaaa tcctaatgca aatcctaatg ccaatccaaa tgcaaatcca 780aatgcaaacc caaacgcaaa ccccaatgca aatcctaatg ccaatccaaa tgcaaatcca 840aatgcaaacc caaacgcaaa ccccaatgca aatcctaatg ccaatccaaa tgcaaatcca 900aatgcaaacc ccaatgcaaa tcctaatgcc aatccaaatg caaatccaaa tgcaaaccca 960aatgcaaacc caaatgcaaa tcctaataaa aacaatcaag gtaatggaca aggtcacaat 1020atgccaaatg acccaaaccg aaatgtagat gaaaatgcta atgccaacaa tgctgtaaaa 1080aataataata acgaagaacc aagtgataag cacatagaac aatatttaaa gaaaatacaa 1140aattctcttt caactgaatg gtccccatgt agtgtaactt gtggaaatgg tattcaagtt 1200agaataaagc ctggctctgc tgataaacct aaagaccaat tagattatga aaatgatatt 1260gaaaaaaaaa tttgtaaaat ggaaaaatgt tccagtgtgt ttaatgtcgt aaatagttca 1320ataggattaa taatggtatt atccttcttg ttccttaatt agataaagaa cacatcttag 1380tttgagttgt acaatattta taaaaatata tactactttt tttcttaatt ttcatttttc 1440tttatatttt cctatttaat ttattttttt gtgaatattt aattatgttt gcgattaatt 1500gtagaaatat atatgtatat actatattta tagaatgtgt tattctcaaa aacaacaaca 1560aaaaaaaaaa aaaaaaaaaa aaaaaagaaa aaaggattaa aagtaaaata gttataaata 1620ttttcaaaaa tatttataac acaaaaaata cttcgaagtt catttaaca 166951388PRTPlasmodium reichenowiMISC_FEATURE(1)..(388)CSP protein GenBank M60972.1 51Met Met Arg Lys Leu Ala Ile Leu Ser Val Ser Ser Phe Leu Phe Val 1 5 10 15 Glu Ala Leu Phe Gln Glu Tyr Gln Cys Tyr Gly Ser Ser Ser Asn Thr 20 25 30 Arg Val Leu Asn Glu Leu Asn Tyr Asp Asn Ala Gly Thr Asn Leu Tyr 35 40 45 Asn Glu Leu Glu Met Asn Tyr Tyr Gly Lys Gln Glu Asn Trp Tyr Ser 50 55 60 Leu Lys Lys Asn Ser Arg Ser Leu Gly Glu Asn Asp Asp Ala Asp Asn 65 70 75 80 Gly Asp Ala Asp Asn Gly Asp Glu Gly Ile Asp Glu Asn Arg Arg His 85 90 95 Arg Asn Lys Glu Gly Lys Glu Lys Leu Lys Lys Pro Lys His Asn Lys 100 105 110 Leu Lys Gln Pro Gly Asn Asp Asn Val Asp Pro Asn Ala Asn Pro Asn 115 120 125 Val Asp Pro Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn 130 135 140 Val Asp Pro Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn 145 150 155 160 Val Asn Pro Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn 165 170 175 Val Asn Pro Asn Ala Asn Pro Asn Val Asn Pro Asn Ala Asn Pro Asn 180 185 190 Val Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 195 200 205 Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 210 215 220 Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 225 230 235 240 Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 245 250 255 Ala Asn Pro Asn Ala Asn Pro Asn Arg Asn Asn Glu Ala Asn Gly Gln 260 265 270 Gly His Asn Lys Pro Asn Asp Gln Asn Arg Asn Val Asn Glu Asn Ala 275 280 285 Asn Ala Asn Asn Ala Gly Arg Asn Asn Asn Asn Glu Glu Pro Ser Asp 290 295 300 Lys His Ile Glu Glu Phe Leu Lys Gln Ile Gln Asn Asn Leu Ser Thr 305 310 315 320 Glu Trp Ser Pro Cys Ser Val Thr Cys Gly Asn Gly Ile Gln Val Arg 325 330 335 Ile Lys Pro Gly Ser Ala Gly Lys Pro Lys Asp Gln Leu Asp Tyr Glu 340 345 350 Asn Asp Leu Glu Lys Lys Ile Cys Lys Met Glu Lys Cys Ser Ser Val 355 360 365 Phe Asn Val Val Asn Ser Ser Ile Gly Leu Ile Met Val Leu Ser Phe 370 375 380 Leu Phe Leu Asn 385 521167DNAPlasmodium

reichenowimisc_feature(1)..(1167)CSP gene GenBank M60972.1 52atgatgagaa aattagctat tttatctgtt tcttcctttt tatttgttga ggccttattc 60caggaatatc agtgctatgg aagttcgtca aacacaaggg ttctaaatga attaaattat 120gataatgcag gcactaattt atataatgaa ttagaaatga attattatgg gaaacaggaa 180aattggtata gccttaaaaa aaatagtaga tcacttggag aaaatgatga tgcagataat 240ggtgatgcag ataatggtga tgaaggtata gatgaaaata gaagacatag aaataaagaa 300ggcaaagaga aattaaagaa accaaaacat aataaattaa agcaaccagg gaatgataat 360gttgatccaa atgccaaccc aaatgtagat ccaaatgcca acccaaatgt agatcccaat 420gcaaacccaa atgtagatcc caatgcaaac ccaaatgtag atcctaatgc aaacccaaat 480gtaaatccca atgcaaaccc aaatgtagat cctaatgcaa acccaaatgt aaatcccaat 540gcaaacccaa atgtaaatcc caatgcaaac ccaaatgtaa atcccaatgc aaacccaaat 600gcaaatccta atgcaaatcc caatgcaaat cccaatgcaa acccaaatgc aaatcctaat 660gcaaatccca atgcaaatcc caatgcaaac ccaaatgcaa atcctaatgc aaatcctaat 720gcaaatccta atgcaaatcc taatgcaaat cctaatgcca atccaaacgc aaacccaaat 780gcaaatccta atagaaacaa tgaagctaat ggacaaggtc acaataagcc aaatgaccaa 840aaccgaaatg taaatgaaaa tgctaatgcc aacaatgctg gaagaaataa taataacgaa 900gaaccaagtg ataagcacat agaagaattt ttaaagcaaa tacaaaataa tctttcaact 960gaatggtccc catgtagtgt aacttgtgga aatggtattc aagttagaat aaagcctggc 1020tctgctggta aacctaaaga ccaattagat tatgaaaatg accttgaaaa aaaaatttgt 1080aaaatggaaa aatgttccag tgtgttcaat gtcgtaaata gttcaatagg attaataatg 1140gtattatcct tcttgttcct taattag 116753367PRTPlasmodium yoeliiMISC_FEATURE(1)..(367)CSP protein GenBank J02695.1 53Met Lys Lys Cys Thr Ile Leu Val Val Ala Ser Leu Leu Leu Val Asp 1 5 10 15 Ser Leu Leu Pro Gly Tyr Gly Gln Asn Lys Ser Val Gln Ala Gln Arg 20 25 30 Asn Leu Asn Glu Leu Cys Tyr Asn Glu Glu Asn Asp Asn Lys Leu Tyr 35 40 45 His Val Leu Asn Ser Lys Asn Gly Lys Ile Tyr Asn Arg Asn Ile Val 50 55 60 Asn Arg Leu Leu Gly Asp Ala Leu Asn Gly Lys Pro Glu Glu Lys Lys 65 70 75 80 Asp Asp Pro Pro Lys Asp Gly Asn Lys Asp Asp Leu Pro Lys Glu Glu 85 90 95 Lys Lys Asp Asp Leu Pro Lys Glu Glu Lys Lys Asp Asp Pro Pro Lys 100 105 110 Asp Pro Lys Lys Asp Asp Pro Pro Lys Glu Ala Gln Asn Lys Leu Asn 115 120 125 Gln Pro Val Val Ala Asp Glu Asn Val Asp Gln Gly Pro Gly Ala Pro 130 135 140 Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly 145 150 155 160 Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly 165 170 175 Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro 180 185 190 Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly 195 200 205 Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro Gln Gly 210 215 220 Pro Gly Ala Pro Gln Glu Pro Pro Gln Gln Pro Pro Gln Gln Pro Pro 225 230 235 240 Gln Gln Pro Pro Gln Gln Pro Pro Gln Gln Pro Pro Gln Gln Pro Pro 245 250 255 Gln Gln Pro Arg Pro Gln Pro Asp Gly Asn Asn Asn Asn Asn Asn Asn 260 265 270 Asn Gly Asn Asn Asn Glu Asp Ser Tyr Val Pro Ser Ala Glu Gln Ile 275 280 285 Leu Glu Phe Val Lys Gln Ile Ser Ser Gln Leu Thr Glu Glu Trp Ser 290 295 300 Gln Cys Ser Val Thr Cys Gly Ser Gly Val Arg Val Arg Lys Arg Lys 305 310 315 320 Asn Val Asn Lys Gln Pro Glu Asn Leu Thr Leu Glu Asp Ile Asp Thr 325 330 335 Glu Ile Cys Lys Met Asp Lys Cys Ser Ser Ile Phe Asn Ile Val Ser 340 345 350 Asn Ser Leu Gly Phe Val Ile Leu Leu Val Leu Val Phe Phe Asn 355 360 365 541580DNAPlasmodium yoeliimisc_feature(1)..(1580)CSP gene GenBank J02695.1 54aaaatgaaga agtgtaccat tttagttgta gcgtcacttt tattagttga ttctctactt 60ccaggatatg gacaaaataa aagtgtccaa gcccaaagaa acttaaacga gctatgttac 120aatgaagaaa atgataataa attgtatcac gtccttaact cgaagaatgg aaaaatatac 180aatcgaaata tagtcaacag attacttggc gatgctctca acggaaaacc agaagaaaaa 240aaagatgatc ccccaaaaga tggcaacaaa gatgatcttc caaaagaaga aaaaaaagat 300gatcttccaa aagaagaaaa aaaagatgat cccccaaaag atcctaaaaa agatgatcca 360ccaaaagagg ctcaaaataa attgaatcaa ccagtagtgg cagatgaaaa tgtagatcaa 420gggccaggag caccacaagg gccaggagca ccacaaggac caggagcacc acagggtcca 480ggagcaccac aaggaccagg agcaccacaa ggaccaggag caccacaagg tccaggagca 540ccacagggtc caggagcacc acagggtcca ggagcaccac aaggaccagg agcaccacag 600gggccaggag caccacaagg accaggagca ccacaaggac caggagcacc acaggggcca 660ggagcaccac aagggccagg agcaccacaa gaaccacccc aacaaccacc ccaacaacca 720ccacaacagc caccacaaca gccaccacaa cagccaccac aacagccacc acaacaacca 780cgcccacagc cagatggtaa taacaacaat aacaataata atggtaataa taatgaagat 840tcttatgtcc caagcgcgga acaaatacta gaatttgtta aacagataag tagtcaactc 900acagaggaat ggtctcaatg tagtgtaacc tgtggttctg gtgtaagagt tagaaaacga 960aaaaatgtaa acaagcaacc agaaaatttg accttagagg atattgatac tgaaatttgt 1020aaaatggata aatgttcaag tatatttaat attgtaagca attcattagg atttgtaata 1080ttattagtat tagtattctt taattaaata aacattacac attattataa atatttatat 1140attatataaa tatttaatat acatataatg tgtgtagact ttattttttg tattgtgaac 1200tttcctcatt tattacgatt atttttatat atatacatat ttaatatgta aattaaaaga 1260aaaaagaaat aatagaaatc ttattatatt tatgatataa attaaaaaaa taaaatatat 1320atacattaca aaatttactt tttttagttt atttttttcg tgtttattat atatgtaatt 1380aacttgttat gacgatatcg aaactttatt tttgagaata tatttttatg aattagaata 1440ttaataatta ttatggttat ttgtttggga atttatataa tttacaatat tatttaaggg 1500caatctaaaa atattttatt gttatgatat ttgaaacatt ttatgtagct atccaaatta 1560tttatttgtg taaaatattt 158055341PRTPlasmodium coateneyiMISC_FEATURE(1)..(341)CSP Protein GenBank AY135360.1 55Met Lys Asn Phe Ile Leu Leu Ala Val Ser Ser Ile Leu Leu Val Asp 1 5 10 15 Leu Phe Pro Thr His Phe Gly His Asn Val Asp Leu Ser Arg Ala Ile 20 25 30 Asn Leu Asn Gly Val Ser Phe Asn Asn Val Asp Thr Ser Leu Leu Gly 35 40 45 Ala Ala Gln Val Arg Gln Ser Ala Ser Arg Gly Arg Gly Leu Gly Glu 50 55 60 Lys Pro Lys Lys Lys Ala Glu Lys Lys Glu Glu Glu Pro Lys Lys Pro 65 70 75 80 Asn Glu Asn Lys Leu Lys Gln Pro Val Asp Gly Ala Arg Asp Gly Pro 85 90 95 Ala Pro Ala Ala Asp Gly Ala Arg Asp Gly Pro Ala Pro Ala Ala Asp 100 105 110 Gly Ala Arg Asp Gly Pro Ala Pro Ala Ala Asp Gly Ala Arg Asp Gly 115 120 125 Pro Ala Pro Ala Ala Asp Gly Ala Arg Asp Gly Pro Ala Pro Ala Ala 130 135 140 Asp Gly Ala Arg Asp Gly Pro Ala Pro Ala Ala Asp Gly Ala Arg Asp 145 150 155 160 Gly Pro Ala Pro Ala Ala Asp Gly Ala Arg Asp Gly Pro Ala Pro Pro 165 170 175 Ala Asp Gly Ala Arg Asp Gly Pro Ala Pro Pro Ala Ala Asp Gly Ala 180 185 190 Arg Asp Gly Pro Ala Pro Pro Ala Ala Asp Gly Ala Arg Asp Gly Pro 195 200 205 Ala Pro Pro Ala Gly Gln Gly Gly Gly Asn Ala Ala Gly Gln Ala Gln 210 215 220 Gly Gly Gly Asn Ala Gly Asn Lys Lys Ala Gly Asp Ala Ala Gly Asn 225 230 235 240 Ala Gly Ala Ala Lys Gly Gln Gly Gln Asn Asn Glu Gly Ala Asn Val 245 250 255 Pro Asn Glu Lys Val Val Asn Asp Tyr Leu Gln Lys Ile Arg Ser Thr 260 265 270 Val Thr Thr Glu Trp Thr Pro Cys Ser Val Thr Cys Gly Asn Gly Val 275 280 285 Arg Leu Arg Arg Lys Ala His Ala Glu Lys Lys Lys Pro Glu Asp Leu 290 295 300 Thr Met Asp Asp Leu Asp Val Glu Val Cys Ala Met Asp Lys Cys Ala 305 310 315 320 Gly Ile Phe Asn Phe Val Ser Asn Ser Leu Gly Leu Val Ile Leu Leu 325 330 335 Val Leu Ala Phe Asn 340 561049DNAPlasmodium coatneyimisc_feature(1)..(1049)CSP gene AY135360.1 56atgaagaact tcattctctt ggctgtttct tccatcctgt tggtggactt gttccccacg 60cacttcggac ataatgtaga tctctccagg gccataaatt taaatggagt aagcttcaat 120aatgtagaca ccagtttact tggcgcagca caggtaagac aaagtgctag ccgaggcaga 180ggacttggtg agaaaccaaa aaaaaaggcg gaaaaaaaag aagaagaacc aaaaaagcca 240aatgaaaata agctgaagca accagtagat ggagcacgag atgggccagc accagcagca 300gatggagcaa gagatggacc agcaccagca gcagatggag cacgagatgg accagcacca 360gcagcagatg gagcaagaga tggaccagca ccagcagcag atggagcaag agatggacca 420gcaccagcag cagatggagc aagagatgga ccagcaccag cagcagatgg agcacgagat 480ggaccagcac cagcagcaga tggagcaaga gatgggccag caccaccagc cgatggagca 540agagatgggc cagcaccacc agcagcagat ggagcacgag atggaccagc accaccagca 600gcagatggag cacgagatgg gccagcacca ccagcaggac aaggaggagg aaatgcagca 660ggccaagcac aaggaggagg aaatgccgga aacaaaaaag caggagacgc agctggaaac 720gcaggagcag caaaaggaca gggacaaaat aatgaaggtg cgaatgtccc aaatgagaaa 780gttgtgaatg attacctaca gaaaattaga tctaccgtta ccaccgaatg gactccatgc 840agtgtaacct gtggaaatgg tgtaagactt agaagaaaag ctcatgcaga aaagaaaaaa 900ccagaggacc ttaccatgga tgaccttgac gtggaagttt gtgcaatgga taagtgcgct 960ggcatattta actttgtgag taattcatta gggctagtca tattgttagt cctagcattc 1020aattaagtag ctgacatcca ttattttcg 104957371PRTPlasmodium knowlesiMISC_FEATURE(1)..(371)CSP protein GenBank DQ350294.2 57Met Arg Asn Phe Ile Leu Leu Ala Val Ser Ser Ile Leu Leu Val Asp 1 5 10 15 Leu Leu Pro Thr His Phe Glu His Asn Val Asp Leu Ser Arg Ala Ile 20 25 30 Asn Val Asn Gly Val Ser Phe Asn Asn Val Asp Thr Ser Ser Leu Gly 35 40 45 Ala Ala Gln Val Arg Gln Ser Ala Ser Arg Gly Arg Gly Leu Gly Glu 50 55 60 Lys Arg Lys Glu Gly Ala Asp Lys Glu Lys Lys Lys Glu Lys Glu Glu 65 70 75 80 Glu Pro Lys Lys Pro Asn Glu Asn Lys Leu Lys Gln Pro Asn Pro Gly 85 90 95 Gln Pro Gln Ala Gln Gly Asp Gly Ala Asn Ala Gly Gln Pro Gln Ala 100 105 110 Gln Gly Asp Gly Ala Asn Ala Gly Gln Pro Gln Ala Gln Gly Asp Gly 115 120 125 Ala Asn Ala Gly Gln Pro Gln Ala Gln Gly Asp Gly Ala Asn Ala Gly 130 135 140 Gln Pro Gln Ala Gln Gly Asp Gly Ala Asn Ala Gly Gln Pro Gln Ala 145 150 155 160 Gln Gly Asp Gly Ala Asn Ala Gly Gln Pro Gln Ala Gln Gly Asp Gly 165 170 175 Ala Asn Ala Gly Gln Pro Gln Ala Gln Gly Asp Gly Ala Asn Ala Gly 180 185 190 Gln Pro Gln Ala Gln Gly Asp Gly Ala Asn Ala Gly Gln Pro Gln Ala 195 200 205 Gln Gly Asp Gly Ala Asn Ala Gly Gln Pro Gln Ala Gln Gly Asp Gly 210 215 220 Ala Asn Ala Gly Gln Pro Gln Ala Gln Gly Asp Arg Ala Asn Ala Gly 225 230 235 240 Gln Pro Gln Ala Gln Gly Asp Gly Ala Asn Val Pro Arg Gln Gly Arg 245 250 255 Asn Gly Gly Gly Ala Pro Ala Gly Gly Asn Glu Gly Asn Lys Gln Ala 260 265 270 Gly Lys Gly Gln Gly Gln Asn Asn Gln Gly Ala Asn Ala Pro Asn Glu 275 280 285 Lys Val Val Asn Asp Tyr Leu His Lys Ile Arg Ser Ser Val Thr Thr 290 295 300 Glu Trp Thr Pro Cys Ser Val Thr Cys Gly Asn Gly Val Arg Ile Arg 305 310 315 320 Arg Lys Ala His Ala Gly Asn Lys Lys Ala Glu Asp Leu Thr Met Asp 325 330 335 Asp Leu Glu Val Glu Ala Cys Val Met Asp Lys Cys Ala Gly Ile Phe 340 345 350 Asn Val Val Ser Asn Ser Leu Gly Leu Val Ile Leu Leu Val Leu Ala 355 360 365 Leu Phe Asn 370 581113DNAPlasmodium knowlesimisc_feature(1)..(1113)CSP gene GenBank DQ350294.2 58atgaggaact tcattctctt ggccgtctcc tccatcctgc tggtggactt gctccccaca 60cacttcgaac ataatgtaga tctctccagg gccataaatg taaatggagt aagcttcaat 120aatgtagaca ccagttcact tggcgcagca caggtaagac aaagtgctag ccgaggcaga 180ggacttggtg agaagcgaaa agaaggagct gataaagaaa agaaaaaaga aaaagaagaa 240gaaccaaaga agccaaatga aaataagctg aaacaaccga atccaggaca accacaagca 300caaggagatg gagcaaatgc aggacaacca caagcacaag gagatggagc aaatgcagga 360caaccacaag cacagggtga tggagcaaat gcaggacaac cacaagcaca aggagatgga 420gcaaatgcag gacaaccaca agcacagggt gatggagcaa atgcaggaca accacaagca 480caaggagatg gagcaaatgc aggacaacca caagcacagg gtgatggagc aaatgcagga 540caaccacaag cacaaggaga tggagcaaat gcaggacaac cacaagcaca gggtgatgga 600gcaaatgcag gacaaccaca agcacagggt gatggagcaa atgcaggaca accacaagca 660cagggtgatg gagcaaatgc aggacaacca caagcacagg gtgatagggc gaatgcagga 720caaccacaag cacaaggaga tggggcaaat gtaccacgac aaggaagaaa cgggggaggt 780gcaccagcag gaggaaatga ggggaataaa caagcaggaa aaggacaggg acaaaacaat 840cagggtgcga atgccccaaa tgaaaaagtt gtgaatgatt acctacacaa aattagatct 900agcgttacca ccgagtggac tccatgcagt gtaacctgtg gaaatggtgt aagaattaga 960agaaaagctc atgcaggtaa taaaaaggca gaggacctta ctatggatga ccttgaggtg 1020gaagcttgtg taatggataa gtgcgctggc atatttaacg ttgtgagtaa ttcattaggg 1080ttagtcatat tgttagtcct agcattattc aat 111359332PRTPlasmodium bergheiMISC_FEATURE(1)..(332)CSP protein GenBank M28887.1 59Met Lys Lys Cys Thr Ile Leu Val Val Ala Ser Leu Leu Leu Val Asn 1 5 10 15 Ser Leu Leu Pro Gly Tyr Gly Gln Asn Lys Ile Ile Gln Ala Gln Arg 20 25 30 Asn Leu Asn Glu Leu Cys Tyr Asn Glu Gly Asn Asp Asn Lys Leu Tyr 35 40 45 His Val Leu Asn Ser Lys Asn Gly Lys Ile Tyr Asn Arg Asn Thr Val 50 55 60 Asn Arg Leu Leu Ala Asp Ala Pro Glu Gly Lys Lys Asn Glu Lys Lys 65 70 75 80 Asn Glu Lys Ile Glu Arg Asn Asn Lys Leu Lys Gln Pro Pro Pro Pro 85 90 95 Pro Asn Pro Asn Asp Pro Pro Pro Pro Asn Pro Asn Asp Pro Pro Pro 100 105 110 Pro Asn Pro Asn Asp Pro Pro Pro Pro Asn Pro Asn Asp Pro Ala Pro 115 120 125 Pro Asn Ala Asn Asp Pro Ala Pro Pro Asn Ala Asn Asp Pro Ala Pro 130 135 140 Pro Asn Ala Asn Asp Pro Ala Pro Pro Asn Ala Asn Asp Pro Ala Pro 145 150 155 160 Pro Asn Ala Asn Asp Pro Ala Pro Pro Asn Ala Asn Asp Pro Ala Pro 165 170 175 Pro Asn Ala Asn Asp Pro Pro Pro Pro Asn Pro Asn Asp Pro Ala Pro 180 185 190 Pro Gln Gly Asn Asn Asn Pro Gln Pro Gln Pro Arg Pro Gln Pro Gln 195 200 205 Pro Gln Pro Gln Pro Gln Pro Gln Pro Gln Pro Gln Pro Gln Pro Arg 210 215 220 Pro Gln Pro Gln Pro Gln Pro Gly Gly Asn Asn Asn Asn Lys Asn Asn 225 230 235 240 Asn Asn Asp Asp Ser Tyr Ile Pro Ser Ala Glu Lys Ile Leu Glu Phe 245 250 255 Val Lys Gln Ile Arg Asp Ser Ile Thr Glu Glu Trp Ser Gln Cys Asn 260 265 270 Val Thr Cys Gly Ser Gly Ile Arg Val Arg Lys Arg Lys Gly Ser Asn 275 280 285 Lys Lys Ala Glu Asp Leu Thr Leu Glu Asp Ile Asp Thr Glu Ile Cys 290 295 300 Lys Met Asp Lys Cys Ser Ser Ile Phe Asn Ile Val Ser Asn Ser Leu 305 310 315 320 Gly Phe Val Ile Leu Leu Val Leu Val Phe Phe Asn 325 330 60999DNAPlasmodium bergheimisc_feature(1)..(999)CSP gene GenBank M28887.1 60atgaagaagt gtaccatttt agttgtagcg tcacttttat tagttaattc tctacttcca 60ggatatggac aaaataaaat catccaagcc caaaggaact taaacgagct atgttacaat 120gaaggaaatg

ataataaatt gtatcacgtg cttaactcta agaatggaaa aatatacaat 180cgaaatacag tcaacagatt acttgccgat gctcccgaag gaaaaaaaaa tgagaaaaaa 240aacgaaaaaa tagagcgtaa taataaattg aaacaaccac caccaccacc aaacccaaat 300gacccaccac caccaaaccc aaatgaccca ccaccaccaa acccaaatga cccaccacca 360ccaaacccaa atgacccagc accaccaaac gcaaatgacc cagcaccacc aaacgcaaat 420gacccagcac caccaaacgc aaatgaccca gcaccaccaa acgcaaatga cccagcacca 480ccaaacgcaa atgacccagc accaccaaac gcaaatgacc cagcaccacc aaacgcaaat 540gacccaccac caccaaaccc aaatgaccca gcaccaccac aaggaaataa caatccacaa 600ccacagccac ggccgcagcc acaaccacag ccacagccac aaccacagcc acagccacaa 660ccacagccac gaccacagcc acaaccacag ccaggtggta ataacaataa caaaaataat 720aataatgacg attcttatat cccaagcgcg gaaaaaatac tagaatttgt taaacagatc 780agggatagta tcacagagga atggtctcaa tgtaacgtaa catgtggttc tggtataaga 840gttagaaaac gaaaaggttc aaataagaaa gcagaagatt tgaccttaga agatattgat 900actgaaattt gtaaaatgga taaatgttca agtatattta atattgtaag caattcatta 960ggatttgtaa tattattagt attagtattc tttaattaa 99961322PRTPlasmodium vivaxMISC_FEATURE(1)..(322)CSP protein GenBank AY674050.1 61Lys Ala Ile Asn Leu Asn Gly Val Asn Phe Asn Asn Val Asp Ala Ser 1 5 10 15 Ser Leu Gly Ala Ala His Val Gly Gln Ser Ala Ser Arg Gly Arg Gly 20 25 30 Leu Gly Glu Asn Pro Asp Asp Glu Glu Gly Asp Ala Lys Lys Lys Lys 35 40 45 Asp Gly Lys Lys Ala Glu Pro Lys Asn Pro Arg Glu Asn Lys Leu Lys 50 55 60 Gln Pro Gly Asp Arg Ala Asp Gly Gln Pro Ala Gly Asp Arg Ala Asp 65 70 75 80 Gly Gln Pro Ala Gly Asp Arg Ala Asp Gly Gln Pro Ala Gly Asp Arg 85 90 95 Ala Ala Gly Gln Pro Ala Gly Asp Arg Ala Asp Gly Gln Pro Ala Gly 100 105 110 Asp Arg Ala Ala Gly Gln Pro Ala Gly Asp Arg Ala Asp Gly Gln Pro 115 120 125 Ala Gly Asp Arg Ala Ala Gly Gln Pro Ala Gly Asp Arg Ala Asp Gly 130 135 140 Gln Pro Ala Gly Asp Arg Ala Ala Gly Gln Pro Ala Gly Asp Arg Ala 145 150 155 160 Asp Gly Gln Pro Ala Gly Asp Arg Ala Ala Gly Gln Pro Ala Gly Asp 165 170 175 Arg Ala Ala Gly Gln Pro Ala Gly Asp Arg Ala Asp Gly Gln Pro Ala 180 185 190 Gly Asp Arg Ala Ala Gly Gln Pro Ala Gly Asp Arg Ala Ala Gly Gln 195 200 205 Pro Ala Gly Asp Arg Ala Ala Gly Gln Pro Ala Gly Asn Gly Ala Gly 210 215 220 Gly Gln Ala Ala Gly Gly Asn Ala Ala Asn Lys Lys Ala Glu Asp Ala 225 230 235 240 Gly Gly Asn Ala Gly Gly Gln Gly Gln Asn Asn Glu Gly Ala Asn Ala 245 250 255 Pro Asn Glu Lys Ser Val Lys Glu Tyr Leu Asp Lys Val Arg Ala Thr 260 265 270 Val Gly Thr Glu Trp Thr Pro Cys Ser Val Thr Cys Gly Val Gly Val 275 280 285 Arg Val Arg Arg Arg Val Asn Ala Ala Asn Lys Lys Pro Glu Asp Leu 290 295 300 Thr Leu Asn Asp Leu Glu Thr Asp Val Cys Thr Met Asp Lys Cys Ala 305 310 315 320 Gly Ile 62968DNAPlasmodium vivaxmisc_feature(1)..(968)CSP gene GenBank AY674050.1 62ccaaggccat aaatttaaat ggagtaaact tcaataatgt agacgccagt tcacttggcg 60cggcacacgt aggacaaagt gctagccgag gcagaggact tggtgagaac ccagatgacg 120aggaaggaga tgctaaaaaa aaaaaggatg gaaagaaagc agaaccaaaa aatccacgtg 180aaaataagct gaaacaacca ggagacagag cagatggaca gccagcagga gacagagcag 240atggacagcc agcaggagac agagcagatg gacagccagc aggtgataga gcagctggac 300aaccagcagg tgatagagca gatggacagc cagcaggcga tagagcagct ggacagccag 360caggcgatag agcagatgga cagccagcag gagatagagc agctggacag ccagcaggcg 420atagagcaga tggacagcca gcaggagata gagcagctgg acagccagca ggcgatagag 480cagatggaca gccagcagga gatagagcag ctggacaacc agcaggtgat agagcagctg 540gacaaccagc aggagataga gcagatggac aaccagcagg agatagagca gctggacagc 600cagcaggaga tagagcagct ggacagccag caggagatag agcagctgga cagccagcag 660gaaatggtgc aggtggacag gcagcaggag gaaatgcggc aaacaagaag gcagaagacg 720caggaggaaa cgcaggagga cagggacaaa ataatgaagg tgcgaatgcc ccaaatgaaa 780agtctgtgaa agaataccta gataaagtta gagctaccgt tggcaccgaa tggactccat 840gcagtgtaac ctgtggagtg ggtgtaagag tcagaagaag agttaatgca gctaacaaaa 900aaccagagga tcttactttg aatgaccttg agactgatgt ttgtacaatg gataagtgtg 960ctggcata 96863429PRTPlasmodium malariaeMISC_FEATURE(1)..(429)CSP protein GenBank J03992.1 63Met Lys Lys Leu Ser Val Leu Ala Ile Ser Ser Phe Leu Ile Val Asp 1 5 10 15 Phe Leu Phe Pro Gly Tyr His His Asn Ser Asn Ser Thr Lys Ser Arg 20 25 30 Asn Leu Ser Glu Leu Cys Tyr Asn Asn Val Asp Thr Lys Leu Phe Asn 35 40 45 Glu Leu Glu Val Arg Tyr Ser Thr Asn Gln Asp His Phe Tyr Asn Tyr 50 55 60 Asn Lys Thr Ile Arg Leu Leu Asn Glu Asn Asn Asn Glu Lys Asp Gly 65 70 75 80 Asn Val Thr Asn Glu Arg Lys Lys Lys Pro Thr Lys Ala Val Glu Asn 85 90 95 Lys Leu Lys Gln Pro Pro Gly Asp Asp Asp Gly Ala Gly Asn Asp Ala 100 105 110 Gly Asn Asp Ala Gly Asn Asp Ala Gly Asn Ala Ala Gly Asn Ala Ala 115 120 125 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 130 135 140 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 145 150 155 160 Gly Asn Asp Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 165 170 175 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Asp Ala Gly Asn Ala Ala 180 185 190 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 195 200 205 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 210 215 220 Gly Asn Ala Ala Gly Asn Asp Ala Gly Asn Ala Ala Gly Asn Ala Ala 225 230 235 240 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 245 250 255 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 260 265 270 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 275 280 285 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Ala Ala 290 295 300 Gly Asn Ala Ala Gly Asn Ala Ala Gly Asn Glu Lys Ala Lys Asn Lys 305 310 315 320 Asp Asn Lys Val Asp Ala Asn Thr Asn Lys Lys Asp Asn Gln Glu Glu 325 330 335 Asn Asn Asp Ser Ser Asn Gly Pro Ser Glu Glu His Ile Lys Asn Tyr 340 345 350 Leu Glu Ser Ile Arg Asn Ser Ile Thr Glu Glu Trp Ser Pro Cys Ser 355 360 365 Val Thr Cys Gly Ser Gly Ile Arg Ala Arg Arg Lys Val Gly Ala Lys 370 375 380 Asn Lys Lys Pro Ala Glu Leu Val Leu Ser Asp Leu Glu Thr Glu Ile 385 390 395 400 Cys Ser Leu Asp Lys Cys Ser Ser Ile Phe Asn Val Val Ser Asn Ser 405 410 415 Leu Gly Ile Val Leu Val Leu Val Leu Ile Leu Phe His 420 425 641545DNAPlasmodium malariae 64tttcacatac ataatttgct gaatattaaa aaaaaataaa taataagtaa ataataaaaa 60cacaaaaaag tatatataaa tatagacttg ctccaacatg aagaagttat ctgtcttagc 120aatatcctct tttttaattg ttgatttcct cttccctgga tatcatcaca actcaaattc 180caccaagtca agaaatttaa gtgagttgtg ttacaataat gtggacacta aattatttaa 240tgagttagaa gtcagatata gcacgaatca agatcatttc tataactata ataagacaat 300cagattactt aatgaaaata acaatgaaaa agatggaaat gtgaccaatg aaagaaaaaa 360aaaacccaca aaagctgttg aaaataaatt gaaacaaccc cccggagatg atgatggcgc 420aggaaatgat gcaggaaatg atgcaggaaa tgatgcagga aatgcagcag gaaatgcagc 480aggaaatgca gcaggaaatg cagcaggtaa cgcagcaggt aacgcagcag gaaatgcagc 540aggaaatgca gcaggtaacg cagcaggaaa tgcagcagga aatgatgcag gaaatgcagc 600aggtaacgca gcaggaaatg cagcaggaaa tgcagcagga aatgcagcag gaaatgatgc 660aggaaatgca gcaggaaatg cagcaggaaa tgcagcaggt aacgcagcag gaaatgcagc 720aggaaatgca gcaggtaacg cagcaggtaa cgcagcagga aatgcagcag gaaatgcagc 780aggaaatgat gcaggaaatg cagcaggtaa cgcagcagga aatgcagcag gaaatgcagc 840aggtaacgca gcaggtaacg cagcaggaaa tgcagcagga aatgcagcag gtaacgcagc 900aggaaatgca gcaggaaatg cagcaggtaa cgcagcaggt aacgcagcag gaaatgcagc 960aggaaatgca gcaggtaacg cagcaggaaa tgcagcagga aatgcagcag gtaacgcagc 1020aggaaatgca gcaggaaatg aaaaagcgaa aaataaggat aataaagtgg atgcaaatac 1080gaataaaaag gacaaccagg aagaaaataa tgattcgtct aatggtccat ctgaagaaca 1140tataaagaat tatttagaaa gtattcgtaa tagtattacg gaggaatggt caccatgtag 1200tgtaacttgt ggaagtggta taagggctag aagaaaggtt ggtgcaaaaa ataagaaacc 1260tgcagaatta gttttaagtg accttgaaac tgaaatttgt tcactagata aatgctccag 1320tatatttaat gtcgtaagta attcgttagg aatagtatta gttttagtct taatactctt 1380tcactaaata aatagcatgt atctttcgaa atattatata catatatatt tatatatatt 1440ttttctttct tttttctttt ttttgtgaat gattactaat gtttgcactt aattgtatat 1500atattatata tattcaatat ataattctaa aaattaccag tattt 154565388PRTPlasmodium gallinaceum 65Met Lys Lys Leu Ala Ile Leu Ser Ala Ser Ser Phe Leu Phe Ala Asp 1 5 10 15 Phe Leu Phe Gln Glu Tyr Gln His Asn Gly Asn Tyr Lys Asn Phe Arg 20 25 30 Leu Leu Asn Glu Val Cys Tyr Asn Asn Met Asn Ile Gln Leu Tyr Asn 35 40 45 Glu Leu Glu Met Glu Asn Tyr Met Ser Asn Thr Tyr Phe Tyr Asn Asn 50 55 60 Lys Lys Thr Ile Arg Leu Leu Gly Glu Asn Asp Asn Glu Ala Asn Val 65 70 75 80 Asn Arg Ala Asn Asn Asn Val Ala Asn Asp Asn Arg Ala Asn Gly Asn 85 90 95 Arg Gly Asn Val Asn Arg Ala Asn Asp Arg Asn Ile Pro Tyr Phe Arg 100 105 110 Glu Asn Val Val Asn Leu Asn Gln Pro Val Gly Gly Asn Gly Gly Val 115 120 125 Gln Pro Ala Gly Gly Asn Gly Gly Val Gln Pro Ala Gly Gly Asn Gly 130 135 140 Gly Val Gln Pro Ala Gly Gly Asn Gly Gly Val Gln Pro Ala Gly Gly 145 150 155 160 Asn Gly Gly Val Gln Pro Ala Gly Gly Asn Gly Gly Val Gln Pro Ala 165 170 175 Gly Gly Asn Gly Gly Val Gln Pro Ala Gly Gly Asn Gly Gly Ala Gln 180 185 190 Pro Val Ala Ala Gly Gly Gly Ala Gln Pro Val Val Ala Asp Gly Gly 195 200 205 Val Gln Pro Leu Arg Gln Glu Gly Asp Ala Glu Glu Asp Gly Gly Asn 210 215 220 Gly Gly Ala Gln Pro Ala Gly Gly Asn Gly Gly Ala Gln Pro Ala Gly 225 230 235 240 Gly Asn Gly Gly Ala Gln Pro Ala Gly Gly Asn Gly Gly Ala Gln Pro 245 250 255 Ala Gly Gly Asn Gly Gly Ala Gln Pro Ala Gly Gly Asn Asp Ala Ala 260 265 270 Lys Pro Asp Gly Gly Asn Asp Asp Asp Lys Pro Glu Gly Gly Asp Glu 275 280 285 Lys Ser Glu Glu Glu Lys Glu Asp Glu Pro Ile Pro Asp Pro Thr Gln 290 295 300 Glu Glu Ile Asp Lys Tyr Leu Lys Ser Ile Leu Gly Asn Val Thr Ser 305 310 315 320 Glu Trp Thr Asn Cys Asn Val Thr Cys Gly Lys Gly Ile Gln Ala Lys 325 330 335 Ile Lys Ser Thr Ser Ala Asn Lys Lys Arg Glu Glu Ile Thr Pro Asn 340 345 350 Asp Val Glu Val Lys Ile Cys Glu Leu Glu Arg Cys Ser Phe Ser Ile 355 360 365 Phe Asn Val Ile Ser Asn Ser Leu Gly Leu Ala Ile Ile Leu Thr Phe 370 375 380 Leu Phe Phe Tyr 385 661260DNAPlasmodium gallinaceummisc_feature(1)..(1260)CSP gene GenBank U65959.1 66tgatttcact aaaaatttta atatatataa tataatagtt taaaatagtg aagaatatat 60ataggtgtac ttcaaaatga agaaattagc cattttatcg gcatcttcgt ttttatttgc 120tgactttcta tttcaagagt atcaacacaa tggaaactac aaaaatttta gacttttaaa 180tgaggtgtgt tataataata tgaatattca attatataat gaattggaaa tggaaaatta 240catgagtaac acatatttct ataataataa aaaaaccatt agattacttg gagaaaatga 300taatgaagca aatgttaata gagcaaataa taatgtagca aatgataata gagcaaatgg 360taatagagga aatgttaata gagcaaatga tagaaatata ccatatttta gagaaaatgt 420tgtgaatctt aatcaaccag ttggaggaaa tggtggtgtt caacctgctg gaggaaatgg 480tggtgttcaa cctgctggag gaaatggtgg tgttcaacct gctggaggta atggtggtgt 540tcaacctgct ggaggaaatg gtggtgttca acctgctgga ggaaatggtg gtgttcaacc 600tgctggaggt aatggtggtg ttcaacctgc tggaggcaat ggtggtgctc aaccagttgc 660agcaggtggt ggtgctcaac cagttgtagc agatggtggt gttcagcctc ttagacaaga 720aggtgatgct gaagaggatg gaggaaatgg tggtgcccaa ccagctggag gaaatggtgg 780tgctcaacca gctggaggaa atggtggtgc tcaaccagct ggaggaaatg gtggtgccca 840acctgctgga ggaaatggtg gtgctcaacc tgctggagga aatgatgctg ctaaacctga 900tggaggaaat gatgatgaca aacctgaagg aggagatgaa aaatctgaag aagaaaagga 960ggatgaacca ataccagatc caactcaaga agaaatagat aaatatttaa aaagcatact 1020tggtaatgtt acatctgaat ggactaattg caatgtaaca tgtgggaaag gtatacaagc 1080taaaataaaa tctacatctg ctaataagaa aagagaagaa attactccaa atgatgttga 1140agtaaaaatt tgcgaactag aaagatgttc ttttagcata tttaatgtta taagcaattc 1200gttaggttta gctataattt taaccttttt atttttttat taaataaata ttataaaatt 126067388PRTPlasmodium reichenowiMISC_FEATURE(1)..(388)CSP protein GenBank U65959.1 67Met Met Arg Lys Leu Ala Ile Leu Ser Val Ser Ser Phe Leu Phe Val 1 5 10 15 Glu Ala Leu Phe Gln Glu Tyr Gln Cys Tyr Gly Ser Ser Ser Asn Thr 20 25 30 Arg Val Leu Asn Glu Leu Asn Tyr Asp Asn Ala Gly Thr Asn Leu Tyr 35 40 45 Asn Glu Leu Glu Met Asn Tyr Tyr Gly Lys Gln Glu Asn Trp Tyr Ser 50 55 60 Leu Lys Lys Asn Ser Arg Ser Leu Gly Glu Asn Asp Asp Ala Asp Asn 65 70 75 80 Gly Asp Ala Asp Asn Gly Asp Glu Gly Ile Asp Glu Asn Arg Arg His 85 90 95 Arg Asn Lys Glu Gly Lys Glu Lys Leu Lys Lys Pro Lys His Asn Lys 100 105 110 Leu Lys Gln Pro Gly Asn Asp Asn Val Asp Pro Asn Ala Asn Pro Asn 115 120 125 Val Asp Pro Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn 130 135 140 Val Asp Pro Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn 145 150 155 160 Val Asn Pro Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn 165 170 175 Val Asn Pro Asn Ala Asn Pro Asn Val Asn Pro Asn Ala Asn Pro Asn 180 185 190 Val Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 195 200 205 Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 210 215 220 Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 225 230 235 240 Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 245 250 255 Ala Asn Pro Asn Ala Asn Pro Asn Arg Asn Asn Glu Ala Asn Gly Gln 260 265 270 Gly His Asn Lys Pro Asn Asp Gln Asn Arg Asn Val Asn Glu Asn Ala 275 280 285 Asn Ala Asn Asn Ala Gly Arg Asn Asn Asn Asn Glu Glu Pro Ser Asp 290 295 300 Lys His Ile Glu Glu Phe Leu Lys Gln Ile Gln Asn Asn Leu Ser Thr 305 310 315 320 Glu Trp Ser Pro Cys Ser Val Thr Cys Gly Asn Gly Ile Gln Val Arg 325 330 335 Ile Lys Pro Gly Ser Ala Gly Lys Pro Lys Asp Gln Leu Asp Tyr Glu 340 345 350 Asn Asp Leu Glu Lys Lys Ile Cys Lys Met Glu Lys Cys Ser Ser Val 355 360 365 Phe Asn Val Val Asn Ser Ser Ile Gly Leu Ile Met

Val Leu Ser Phe 370 375 380 Leu Phe Leu Asn 385 681167DNAPlasmodium reichenowi 68atgatgagaa aattagctat tttatctgtt tcttcctttt tatttgttga ggccttattc 60caggaatatc agtgctatgg aagttcgtca aacacaaggg ttctaaatga attaaattat 120gataatgcag gcactaattt atataatgaa ttagaaatga attattatgg gaaacaggaa 180aattggtata gccttaaaaa aaatagtaga tcacttggag aaaatgatga tgcagataat 240ggtgatgcag ataatggtga tgaaggtata gatgaaaata gaagacatag aaataaagaa 300ggcaaagaga aattaaagaa accaaaacat aataaattaa agcaaccagg gaatgataat 360gttgatccaa atgccaaccc aaatgtagat ccaaatgcca acccaaatgt agatcccaat 420gcaaacccaa atgtagatcc caatgcaaac ccaaatgtag atcctaatgc aaacccaaat 480gtaaatccca atgcaaaccc aaatgtagat cctaatgcaa acccaaatgt aaatcccaat 540gcaaacccaa atgtaaatcc caatgcaaac ccaaatgtaa atcccaatgc aaacccaaat 600gcaaatccta atgcaaatcc caatgcaaat cccaatgcaa acccaaatgc aaatcctaat 660gcaaatccca atgcaaatcc caatgcaaac ccaaatgcaa atcctaatgc aaatcctaat 720gcaaatccta atgcaaatcc taatgcaaat cctaatgcca atccaaacgc aaacccaaat 780gcaaatccta atagaaacaa tgaagctaat ggacaaggtc acaataagcc aaatgaccaa 840aaccgaaatg taaatgaaaa tgctaatgcc aacaatgctg gaagaaataa taataacgaa 900gaaccaagtg ataagcacat agaagaattt ttaaagcaaa tacaaaataa tctttcaact 960gaatggtccc catgtagtgt aacttgtgga aatggtattc aagttagaat aaagcctggc 1020tctgctggta aacctaaaga ccaattagat tatgaaaatg accttgaaaa aaaaatttgt 1080aaaatggaaa aatgttccag tgtgttcaat gtcgtaaata gttcaatagg attaataatg 1140gtattatcct tcttgttcct taattag 116769200DNAArtificialDNA FLAP of CAEV 69gttccagcca caatttgtcg ctgtagaatc agccatagca gcagccctag tcgccataaa 60tataaaaaga aagggtgggc tggggacaag ccctatggat atttttatat ataataaaga 120acagaaaaga ataaataata aatataataa aaattctcaa aaaattcaat tctgttatta 180cagaataagg aaaagaggac 20070200DNAArtificialDNA FLAP of EIAV 70cttgtaacaa agggagggaa agtatgggag gacagacacc atgggaagta tttatcacta 60atcaagcaca agtaatacat gagaaacttt tactacagca agcacaatcc tccaaaaaat 120tttgttttta caaaatccct ggtgaacatg attggaaggg acctactagg gtgctgtgga 180agggtgatgg tgcagtagta 20071200DNAArtificialDNA FLAP of VISNA 71ggaccctcat tactctaaat ataaaaagaa agggtgggct agggacaagc cctatggata 60tatttatatt taataaggaa caacaaagaa tacagcaaca aagtaaatca aaacaagaaa 120aaattcgatt ttgttattac agaacaagaa aaagagggca tccaggagag tggcaaggac 180caacacaggt actttggggc 20072200DNAArtificialDNA FLAP of VISNA 72tactgatggc ttgcatactt cacaatttta aaagaaaggg aggaataggg ggacagactt 60cagcagagag actaattaat ataataacaa cacaattaga aatacaacat ttacaaacca 120aaattcaaaa aattttaaat tttagagtct actacagaga agggagagac cctgtgtgga 180aaggaccggc acaattaatc 20073200DNAArtificialDNA FLAP of HIV-2 ROD 73tgcatgaatt ttaaaagaag ggggggaata ggggatatga ctccatcaga aagattaatc 60aatatgatca ccacagaaca agagatacaa ttcctccaag ccaaaaattc aaaattaaaa 120gattttcggg tctatttcag agaaggcaga gatcagttgt ggaaaggacc tggggaacta 180ctgtggaaag gagaaggagc 20074200DNAArtificialDNA FLAP of HIV-1 LAI 74cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa 60gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa 120aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg aaaggaccag 180caaagctcct ctggaaaggt 20075119DNAArtificialDNA FLAP of HIV-1 75ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat 60agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttc 119761578DNAArtificialVSV-G Indiana optimized 76ctcggatcct gatcagccac catgaaatgc ctgctctatc tggccttcct ctttatcggc 60gtgaactgta agttcacgat cgtgtttccc cacaatcaga agggaaactg gaagaacgtc 120ccgagcaact accactactg ccctagctca agcgacctga actggcacaa cgacctgatc 180ggcaccgcta tccaggtgaa gatgccaaag agccacaagg ccatccaagc cgacggctgg 240atgtgtcacg ccagcaaatg ggtgacgacg tgcgattttc gctggtatgg ccccaagtac 300atcacccaat caatccgctc atttacaccc agcgtggagc aatgtaagga gagcatcgag 360cagaccaagc aggggacctg gctcaacccc ggcttcccac cgcaaagctg cggatacgcc 420accgtgaccg acgctgaggc cgtcatcgtg caggtgaccc cgcaccacgt gctggtggac 480gagtacaccg gcgagtgggt ggattcacag tttatcaacg gaaagtgtag caattacatc 540tgccccaccg tgcacaacag caccacctgg cactcagact ataaggtgaa gggcctctgc 600gacagcaatc tgatctcaat ggacatcacc ttctttagcg aagacggcga actctcaagc 660ctcgggaagg aaggcaccgg gttccgcagc aattactttg cttacgaaac cggcggcaag 720gcctgcaaga tgcaatactg caagcactgg ggcgtgcgcc tgccaagcgg cgtgtggttt 780gagatggctg ataaggacct gttcgccgct gcccgcttcc cggaatgccc cgaggggagc 840agcatcagcg cccccagcca gacatcagtg gacgtgagcc tgatccagga tgtggaacgc 900atcctggact acagcctgtg tcaggaaacg tggagcaaga tccgcgccgg actgcctatc 960agccccgtgg atctcagcta cctggcccca aagaacccag gcaccggacc cgcctttaca 1020atcatcaacg gcaccctgaa gtactttgaa acacgctaca tccgcgtcga catcgccgct 1080cccatcctct cacgcatggt gggcatgatc tcagggacga ccacggagcg cgagctgtgg 1140gatgactggg ccccgtatga agatgtggag atcggaccta acggcgtgct gcgcacatca 1200agcgggtaca agttcccgct gtacatgatc ggccacggca tgctggacag cgacctgcac 1260ctcagctcaa aggcccaggt ctttgagcac ccacacatcc aggacgctgc cagccagctc 1320cccgacgacg aaagcctgtt ctttggagat acagggctca gcaagaaccc catcgagctg 1380gtcgagggct ggttctcaag ctggaagagc agcatcgctt catttttttt catcatcggc 1440ctcatcatcg ggctgtttct ggtgctgcgc gtcggcatcc acctgtgcat caagctgaag 1500cacaccaaga agcgccagat ctataccgac atcgagatga atcgcctggg gaagtaagaa 1560ttctgcagat atccagca 157877511PRTArtificialVSV-G Indiana 77Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys 1 5 10 15 Lys Phe Thr Ile Val Phe Pro His Asn Gln Lys Gly Asn Trp Lys Asn 20 25 30 Val Pro Ser Asn Tyr His Tyr Cys Pro Ser Ser Ser Asp Leu Asn Trp 35 40 45 His Asn Asp Leu Ile Gly Thr Ala Ile Gln Val Lys Met Pro Lys Ser 50 55 60 His Lys Ala Ile Gln Ala Asp Gly Trp Met Cys His Ala Ser Lys Trp 65 70 75 80 Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile Thr Gln 85 90 95 Ser Ile Arg Ser Phe Thr Pro Ser Val Glu Gln Cys Lys Glu Ser Ile 100 105 110 Glu Gln Thr Lys Gln Gly Thr Trp Leu Asn Pro Gly Phe Pro Pro Gln 115 120 125 Ser Cys Gly Tyr Ala Thr Val Thr Asp Ala Glu Ala Val Ile Val Gln 130 135 140 Val Thr Pro His His Val Leu Val Asp Glu Tyr Thr Gly Glu Trp Val 145 150 155 160 Asp Ser Gln Phe Ile Asn Gly Lys Cys Ser Asn Tyr Ile Cys Pro Thr 165 170 175 Val His Asn Ser Thr Thr Trp His Ser Asp Tyr Lys Val Lys Gly Leu 180 185 190 Cys Asp Ser Asn Leu Ile Ser Met Asp Ile Thr Phe Phe Ser Glu Asp 195 200 205 Gly Glu Leu Ser Ser Leu Gly Lys Glu Gly Thr Gly Phe Arg Ser Asn 210 215 220 Tyr Phe Ala Tyr Glu Thr Gly Gly Lys Ala Cys Lys Met Gln Tyr Cys 225 230 235 240 Lys His Trp Gly Val Arg Leu Pro Ser Gly Val Trp Phe Glu Met Ala 245 250 255 Asp Lys Asp Leu Phe Ala Ala Ala Arg Phe Pro Glu Cys Pro Glu Gly 260 265 270 Ser Ser Ile Ser Ala Pro Ser Gln Thr Ser Val Asp Val Ser Leu Ile 275 280 285 Gln Asp Val Glu Arg Ile Leu Asp Tyr Ser Leu Cys Gln Glu Thr Trp 290 295 300 Ser Lys Ile Arg Ala Gly Leu Pro Ile Ser Pro Val Asp Leu Ser Tyr 305 310 315 320 Leu Ala Pro Lys Asn Pro Gly Thr Gly Pro Ala Phe Thr Ile Ile Asn 325 330 335 Gly Thr Leu Lys Tyr Phe Glu Thr Arg Tyr Ile Arg Val Asp Ile Ala 340 345 350 Ala Pro Ile Leu Ser Arg Met Val Gly Met Ile Ser Gly Thr Thr Thr 355 360 365 Glu Arg Glu Leu Trp Asp Asp Trp Ala Pro Tyr Glu Asp Val Glu Ile 370 375 380 Gly Pro Asn Gly Val Leu Arg Thr Ser Ser Gly Tyr Lys Phe Pro Leu 385 390 395 400 Tyr Met Ile Gly His Gly Met Leu Asp Ser Asp Leu His Leu Ser Ser 405 410 415 Lys Ala Gln Val Phe Glu His Pro His Ile Gln Asp Ala Ala Ser Gln 420 425 430 Leu Pro Asp Asp Glu Ser Leu Phe Phe Gly Asp Thr Gly Leu Ser Lys 435 440 445 Asn Pro Ile Glu Leu Val Glu Gly Trp Phe Ser Ser Trp Lys Ser Ser 450 455 460 Ile Ala Ser Phe Phe Phe Ile Ile Gly Leu Ile Ile Gly Leu Phe Leu 465 470 475 480 Val Leu Arg Val Gly Ile His Leu Cys Ile Lys Leu Lys His Thr Lys 485 490 495 Lys Arg Gln Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys 500 505 510 781597DNAArtificialVSV-G New Jersey optimized 78taccgagctc ggatcctgat cagccaccat gctgtcatat ctgatctttg ccctggctgt 60gagcccaatc ctcggaaaga tcgaaatcgt gttcccacaa cacaccacag gggactggaa 120gcgcgtgccc cacgagtaca actactgccc gacctcagcc gacaagaata gccacggcac 180gcagaccggc atccctgtgg agctgaccat gcccaagggg ctcacaacgc accaagtcga 240aggcttcatg tgccacagcg ctctctggat gacaacctgc gattttcgct ggtatggccc 300caagtacatc acgcacagca tccacaatga ggaaccaacc gactaccagt gcctcgaagc 360catcaagtca tacaaggatg gggtgagctt caaccccggc ttcccgcccc aatcatgtgg 420ctacggcacc gtgaccgacg ccgaggccca catcgtgacc gtgacacccc actcagtcaa 480ggtggacgag tacacaggcg aatggatcga cccccacttc atcgggggcc gctgtaaggg 540ccaaatctgc gagaccgtgc acaacagcac caagtggttt acgtcatcag acggcgaaag 600cgtgtgcagc caactgttta cgctcgtggg cggcatcttc tttagcgaca gcgaggagat 660caccagcatg ggcctcccgg agacaggaat ccgcagcaac tactttccgt acatcagcac 720cgagggaatc tgtaagatgc ctttttgccg caagcaggga tataagctga agaatgacct 780gtggttccag atcatggacc cggacctgga caagaccgtc cgcgatctgc cccacatcaa 840ggactgtgat ctgtcatcaa gcatcatcac ccccggagaa cacgccacgg acatcagcct 900catcagcgat gtggagcgca tcctcgacta cgctctctgc cagaacacat ggagcaagat 960cgaaagcggc gaacccatca ccccagtgga cctgagctat ctcggcccaa agaaccccgg 1020cgtggggccc gtgttcacca tcatcaacgg gagcctgcac tactttacaa gcaagtatct 1080gcgcgtggag ctcgaaagcc cagtcatccc ccgcatggag gggaaggtgg ccgggacccg 1140catcgtgcgc cagctgtggg accagtggtt cccttttggc gaggtggaaa tcggccccaa 1200cggcgtgctg aagaccaagc aaggatataa gttcccgctg cacatcatcg ggacgggcga 1260agtggacagc gatatcaaga tggagcgcgt ggtcaagcac tgggagcacc cacacatcga 1320ggctgctcag acctttctca agaaggacga taccggcgaa gtcctgtatt acggggatac 1380gggagtgagc aagaaccctg tggagctggt ggaaggctgg ttcagcggat ggcgctcaag 1440cctgatgggc gtgctggccg tcatcatcgg atttgtgatc ctgatgttcc tcatcaagct 1500gatcggcgtg ctgtcaagcc tgttccgccc taagcgccgc ccaatctaca agagcgacgt 1560cgagatggcc cactttcgct aagaattctg cagatat 1597791563DNAArtificialfusion VSV-G Ghandipura / Indiana 79atg acc agc agc gtg acc atc agc gtg gtg ctg ctg atc agc ttc atc 48Met Thr Ser Ser Val Thr Ile Ser Val Val Leu Leu Ile Ser Phe Ile 1 5 10 15 acc ccc ctg tac agc tac ctg agc att gcc ttc ccc gag aac acc aag 96Thr Pro Leu Tyr Ser Tyr Leu Ser Ile Ala Phe Pro Glu Asn Thr Lys 20 25 30 ctg gac tgg aag ccc gtg acc aag aac acc cgg tac tgc ccc atg ggc 144Leu Asp Trp Lys Pro Val Thr Lys Asn Thr Arg Tyr Cys Pro Met Gly 35 40 45 ggc gag tgg ttt ctg gaa ccc ggc ctg cag gaa gag agc ttc ctg agc 192Gly Glu Trp Phe Leu Glu Pro Gly Leu Gln Glu Glu Ser Phe Leu Ser 50 55 60 agc acc ccc atc ggc gcc acc ccc agc aag agc gac ggc ttc ctg tgc 240Ser Thr Pro Ile Gly Ala Thr Pro Ser Lys Ser Asp Gly Phe Leu Cys 65 70 75 80 cac gcc gcc aag tgg gtg acc acc tgc gac ttc cgg tgg tac ggc ccc 288His Ala Ala Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro 85 90 95 aag tac atc acc cac agc atc cac aac atc aag ccc acc aga agc gac 336Lys Tyr Ile Thr His Ser Ile His Asn Ile Lys Pro Thr Arg Ser Asp 100 105 110 tgc gac aca gcc ctg gcc tct tac aag agc ggc acc ctg gtg tcc ctg 384Cys Asp Thr Ala Leu Ala Ser Tyr Lys Ser Gly Thr Leu Val Ser Leu 115 120 125 ggc ttc cct ccc gag agc tgc ggc tac gcc agc gtg acc gac agc gag 432Gly Phe Pro Pro Glu Ser Cys Gly Tyr Ala Ser Val Thr Asp Ser Glu 130 135 140 ttc ctg gtg att atg att acc ccc cac cac gtg ggc gtg gac gac tac 480Phe Leu Val Ile Met Ile Thr Pro His His Val Gly Val Asp Asp Tyr 145 150 155 160 cgg ggc cac tgg gtg gac cct ctg ttc gtg gga ggg gaa tgc gac cag 528Arg Gly His Trp Val Asp Pro Leu Phe Val Gly Gly Glu Cys Asp Gln 165 170 175 agc tac tgc gat acc atc cac aac tcc agc gtg tgg att ccc gcc gac 576Ser Tyr Cys Asp Thr Ile His Asn Ser Ser Val Trp Ile Pro Ala Asp 180 185 190 cag acc aag aag aac atc tgc ggc cag agc ttc acc cct ctg acc gtg 624Gln Thr Lys Lys Asn Ile Cys Gly Gln Ser Phe Thr Pro Leu Thr Val 195 200 205 acc gtg gcc tac gac aag acc aaa gag att gcc gcc gga ggg atc gtg 672Thr Val Ala Tyr Asp Lys Thr Lys Glu Ile Ala Ala Gly Gly Ile Val 210 215 220 ttc aag agc aag tac cac agc cac atg gaa ggc gcc agg acc tgc aga 720Phe Lys Ser Lys Tyr His Ser His Met Glu Gly Ala Arg Thr Cys Arg 225 230 235 240 ctg tcc tac tgc ggc cgg aac ggc atc aag ttc ccc aac ggc gag tgg 768Leu Ser Tyr Cys Gly Arg Asn Gly Ile Lys Phe Pro Asn Gly Glu Trp 245 250 255 gtg tcc ctg atg ctg aag ctg cgg agc aag cgg aac ctg tac ttc ccc 816Val Ser Leu Met Leu Lys Leu Arg Ser Lys Arg Asn Leu Tyr Phe Pro 260 265 270 tgc ctg aag atg tgc ccc acc ggc atc cgg ggc gag atc tac ccc agc 864Cys Leu Lys Met Cys Pro Thr Gly Ile Arg Gly Glu Ile Tyr Pro Ser 275 280 285 atc aga tgg gcc cag gtg ctg acc agc gag atc cag aga atc ctg gac 912Ile Arg Trp Ala Gln Val Leu Thr Ser Glu Ile Gln Arg Ile Leu Asp 290 295 300 tac agc ctg tgc cag aac acc tgg gac aag gtg gag cgg aaa gag ccc 960Tyr Ser Leu Cys Gln Asn Thr Trp Asp Lys Val Glu Arg Lys Glu Pro 305 310 315 320 ctg agc ccc ctg gac ctg agc tac ctg gcc agc aag tcc ccc ggc aag 1008Leu Ser Pro Leu Asp Leu Ser Tyr Leu Ala Ser Lys Ser Pro Gly Lys 325 330 335 ggc ctg gcc tac acc gtg atc aac ggc acc ctg agc ttc gcc cac acc 1056Gly Leu Ala Tyr Thr Val Ile Asn Gly Thr Leu Ser Phe Ala His Thr 340 345 350 aga tac gtg cgg atg tgg atc gac ggc ccc gtg ctg aaa gag ccc aag 1104Arg Tyr Val Arg Met Trp Ile Asp Gly Pro Val Leu Lys Glu Pro Lys 355 360 365 ggc aag aga gag agc ccc agc ggc atc agc agc gac atc tgg acc cag 1152Gly Lys Arg Glu Ser Pro Ser Gly Ile Ser Ser Asp Ile Trp Thr Gln 370 375 380 tgg ttc aag tac ggc gac atg gaa atc ggc ccc aac ggc ctg ctg aaa 1200Trp Phe Lys Tyr Gly Asp Met Glu Ile Gly Pro Asn Gly Leu Leu Lys 385 390 395 400 aca gcc ggc gga tac aag ttt cct tgg cac ctg atc ggc atg ggc atc 1248Thr Ala Gly Gly Tyr Lys Phe Pro Trp His Leu Ile Gly Met Gly Ile 405 410 415 gtg gac aac gag ctg cac gag ctg tcc gag gcc aac ccc ctg gat cac 1296Val Asp Asn Glu Leu His Glu Leu Ser Glu Ala Asn Pro Leu Asp His 420 425 430 ccc cag ctg ccc cac gcc cag agc att gcc gac gac agc gag gaa atc 1344Pro Gln Leu Pro His Ala Gln Ser Ile Ala Asp Asp Ser Glu Glu Ile 435 440 445 ttc ttc ggc gac acc ggc gtg agc aag aac ccc gtg gaa ctg gtg aca 1392Phe Phe Gly Asp Thr Gly Val Ser Lys Asn Pro Val Glu Leu Val Thr 450 455 460

ggc tgg ttc acc agc tgg aaa agc agc atc gct tca ttt ttt ttc atc 1440Gly Trp Phe Thr Ser Trp Lys Ser Ser Ile Ala Ser Phe Phe Phe Ile 465 470 475 480 atc ggc ctc atc atc ggg ctg ttt ctg gtg ctg cgc gtc ggc atc cac 1488Ile Gly Leu Ile Ile Gly Leu Phe Leu Val Leu Arg Val Gly Ile His 485 490 495 ctg tgc atc aag ctg aag cac acc aag aag cgc cag atc tat acc gac 1536Leu Cys Ile Lys Leu Lys His Thr Lys Lys Arg Gln Ile Tyr Thr Asp 500 505 510 atc gag atg aat cgc ctg ggg aag taa 1563Ile Glu Met Asn Arg Leu Gly Lys 515 520 80520PRTArtificialSynthetic Construct 80Met Thr Ser Ser Val Thr Ile Ser Val Val Leu Leu Ile Ser Phe Ile 1 5 10 15 Thr Pro Leu Tyr Ser Tyr Leu Ser Ile Ala Phe Pro Glu Asn Thr Lys 20 25 30 Leu Asp Trp Lys Pro Val Thr Lys Asn Thr Arg Tyr Cys Pro Met Gly 35 40 45 Gly Glu Trp Phe Leu Glu Pro Gly Leu Gln Glu Glu Ser Phe Leu Ser 50 55 60 Ser Thr Pro Ile Gly Ala Thr Pro Ser Lys Ser Asp Gly Phe Leu Cys 65 70 75 80 His Ala Ala Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro 85 90 95 Lys Tyr Ile Thr His Ser Ile His Asn Ile Lys Pro Thr Arg Ser Asp 100 105 110 Cys Asp Thr Ala Leu Ala Ser Tyr Lys Ser Gly Thr Leu Val Ser Leu 115 120 125 Gly Phe Pro Pro Glu Ser Cys Gly Tyr Ala Ser Val Thr Asp Ser Glu 130 135 140 Phe Leu Val Ile Met Ile Thr Pro His His Val Gly Val Asp Asp Tyr 145 150 155 160 Arg Gly His Trp Val Asp Pro Leu Phe Val Gly Gly Glu Cys Asp Gln 165 170 175 Ser Tyr Cys Asp Thr Ile His Asn Ser Ser Val Trp Ile Pro Ala Asp 180 185 190 Gln Thr Lys Lys Asn Ile Cys Gly Gln Ser Phe Thr Pro Leu Thr Val 195 200 205 Thr Val Ala Tyr Asp Lys Thr Lys Glu Ile Ala Ala Gly Gly Ile Val 210 215 220 Phe Lys Ser Lys Tyr His Ser His Met Glu Gly Ala Arg Thr Cys Arg 225 230 235 240 Leu Ser Tyr Cys Gly Arg Asn Gly Ile Lys Phe Pro Asn Gly Glu Trp 245 250 255 Val Ser Leu Met Leu Lys Leu Arg Ser Lys Arg Asn Leu Tyr Phe Pro 260 265 270 Cys Leu Lys Met Cys Pro Thr Gly Ile Arg Gly Glu Ile Tyr Pro Ser 275 280 285 Ile Arg Trp Ala Gln Val Leu Thr Ser Glu Ile Gln Arg Ile Leu Asp 290 295 300 Tyr Ser Leu Cys Gln Asn Thr Trp Asp Lys Val Glu Arg Lys Glu Pro 305 310 315 320 Leu Ser Pro Leu Asp Leu Ser Tyr Leu Ala Ser Lys Ser Pro Gly Lys 325 330 335 Gly Leu Ala Tyr Thr Val Ile Asn Gly Thr Leu Ser Phe Ala His Thr 340 345 350 Arg Tyr Val Arg Met Trp Ile Asp Gly Pro Val Leu Lys Glu Pro Lys 355 360 365 Gly Lys Arg Glu Ser Pro Ser Gly Ile Ser Ser Asp Ile Trp Thr Gln 370 375 380 Trp Phe Lys Tyr Gly Asp Met Glu Ile Gly Pro Asn Gly Leu Leu Lys 385 390 395 400 Thr Ala Gly Gly Tyr Lys Phe Pro Trp His Leu Ile Gly Met Gly Ile 405 410 415 Val Asp Asn Glu Leu His Glu Leu Ser Glu Ala Asn Pro Leu Asp His 420 425 430 Pro Gln Leu Pro His Ala Gln Ser Ile Ala Asp Asp Ser Glu Glu Ile 435 440 445 Phe Phe Gly Asp Thr Gly Val Ser Lys Asn Pro Val Glu Leu Val Thr 450 455 460 Gly Trp Phe Thr Ser Trp Lys Ser Ser Ile Ala Ser Phe Phe Phe Ile 465 470 475 480 Ile Gly Leu Ile Ile Gly Leu Phe Leu Val Leu Arg Val Gly Ile His 485 490 495 Leu Cys Ile Lys Leu Lys His Thr Lys Lys Arg Gln Ile Tyr Thr Asp 500 505 510 Ile Glu Met Asn Arg Leu Gly Lys 515 520 811539DNAArtificialfusion VSV-G Cocal / Indiana 81atg aac ttt ctg ctg ctg aca ttc atc gtg ctg cct ctg tgc agc cac 48Met Asn Phe Leu Leu Leu Thr Phe Ile Val Leu Pro Leu Cys Ser His 1 5 10 15 gcc aag ttc agc atc gtg ttc ccc cag agc cag aag ggc aac tgg aag 96Ala Lys Phe Ser Ile Val Phe Pro Gln Ser Gln Lys Gly Asn Trp Lys 20 25 30 aac gtg ccc agc agc tac cac tac tgc ccc agc agc agc gac cag aac 144Asn Val Pro Ser Ser Tyr His Tyr Cys Pro Ser Ser Ser Asp Gln Asn 35 40 45 tgg cac aac gac ctg ctg ggc atc acc atg aag gtg aaa atg ccc aag 192Trp His Asn Asp Leu Leu Gly Ile Thr Met Lys Val Lys Met Pro Lys 50 55 60 acc cac aag gcc att cag gct gac ggc tgg atg tgc cac gcc gcc aag 240Thr His Lys Ala Ile Gln Ala Asp Gly Trp Met Cys His Ala Ala Lys 65 70 75 80 tgg atc acc acc tgc gac ttc cgg tgg tac ggc ccc aag tac atc acc 288Trp Ile Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile Thr 85 90 95 cac agc atc cac tcc atc cag ccc acc tcc gag cag tgc aaa gag agc 336His Ser Ile His Ser Ile Gln Pro Thr Ser Glu Gln Cys Lys Glu Ser 100 105 110 atc aag cag acc aag cag ggc acc tgg atg agc ccc ggc ttc cca ccc 384Ile Lys Gln Thr Lys Gln Gly Thr Trp Met Ser Pro Gly Phe Pro Pro 115 120 125 cag aac tgc ggc tac gcc acc gtg acc gac agc gtg gcc gtg gtg gtg 432Gln Asn Cys Gly Tyr Ala Thr Val Thr Asp Ser Val Ala Val Val Val 130 135 140 cag gcc acc ccc cac cac gtg ctg gtc gac gag tac acc ggc gag tgg 480Gln Ala Thr Pro His His Val Leu Val Asp Glu Tyr Thr Gly Glu Trp 145 150 155 160 atc gac agc cag ttc ccc aac ggc aag tgc gag aca gag gaa tgc gag 528Ile Asp Ser Gln Phe Pro Asn Gly Lys Cys Glu Thr Glu Glu Cys Glu 165 170 175 aca gtg cac aac agc acc gtg tgg tac agc gac tac aag gtg acc ggc 576Thr Val His Asn Ser Thr Val Trp Tyr Ser Asp Tyr Lys Val Thr Gly 180 185 190 ctg tgc gac gcc acc ctg gtg gac acc gag atc acc ttt ttc agc gag 624Leu Cys Asp Ala Thr Leu Val Asp Thr Glu Ile Thr Phe Phe Ser Glu 195 200 205 gac ggc aag aaa gag tcc atc ggc aag ccc aac acc ggc tac aga agc 672Asp Gly Lys Lys Glu Ser Ile Gly Lys Pro Asn Thr Gly Tyr Arg Ser 210 215 220 aac tac ttc gcc tac gag aag ggc gac aaa gtg tgc aag atg aac tac 720Asn Tyr Phe Ala Tyr Glu Lys Gly Asp Lys Val Cys Lys Met Asn Tyr 225 230 235 240 tgc aag cat gcc gga gtg agg ctg cct agc ggc gtg tgg ttc gag ttc 768Cys Lys His Ala Gly Val Arg Leu Pro Ser Gly Val Trp Phe Glu Phe 245 250 255 gtg gac cag gac gtg tac gcc gcc gcc aag ctg ccc gag tgc ccc gtg 816Val Asp Gln Asp Val Tyr Ala Ala Ala Lys Leu Pro Glu Cys Pro Val 260 265 270 ggc gcc acc atc agc gcc ccc acc cag acc agc gtg gac gtg agc ctg 864Gly Ala Thr Ile Ser Ala Pro Thr Gln Thr Ser Val Asp Val Ser Leu 275 280 285 atc ctg gac gtg gag aga atc ctg gac tac tct ctg tgt cag gaa acc 912Ile Leu Asp Val Glu Arg Ile Leu Asp Tyr Ser Leu Cys Gln Glu Thr 290 295 300 tgg tcc aag atc aga tcc aag cag ccc gtg agc cct gtg gac ctg agc 960Trp Ser Lys Ile Arg Ser Lys Gln Pro Val Ser Pro Val Asp Leu Ser 305 310 315 320 tac ctg gcc cct aag aac ccc ggc acc ggc cct gcc ttc acc atc atc 1008Tyr Leu Ala Pro Lys Asn Pro Gly Thr Gly Pro Ala Phe Thr Ile Ile 325 330 335 aac ggc acc ctg aag tac ttc gag aca cgg tac atc cgg atc gac atc 1056Asn Gly Thr Leu Lys Tyr Phe Glu Thr Arg Tyr Ile Arg Ile Asp Ile 340 345 350 gac aac ccc atc atc agc aag atg gtg ggc aag atc agc ggc agc cag 1104Asp Asn Pro Ile Ile Ser Lys Met Val Gly Lys Ile Ser Gly Ser Gln 355 360 365 acc gag cgg gag ctg tgg acc gag tgg ttc ccc tac gag ggc gtg gag 1152Thr Glu Arg Glu Leu Trp Thr Glu Trp Phe Pro Tyr Glu Gly Val Glu 370 375 380 atc ggc ccc aat ggc atc ctg aaa acc cct acc ggc tac aag ttc ccc 1200Ile Gly Pro Asn Gly Ile Leu Lys Thr Pro Thr Gly Tyr Lys Phe Pro 385 390 395 400 ctg ttc atg atc ggc cac ggc atg ctg gac agc gac ctg cac aag acc 1248Leu Phe Met Ile Gly His Gly Met Leu Asp Ser Asp Leu His Lys Thr 405 410 415 tcc cag gcc gag gtg ttc gag cac ccc cac ctg gcc gag gcc ccc aag 1296Ser Gln Ala Glu Val Phe Glu His Pro His Leu Ala Glu Ala Pro Lys 420 425 430 cag ctg ccc gaa gag gaa acc ctg ttc ttc ggc gac acc ggc atc tcc 1344Gln Leu Pro Glu Glu Glu Thr Leu Phe Phe Gly Asp Thr Gly Ile Ser 435 440 445 aag aac cct gtg gag ctg atc gag ggc tgg ttc agc agc tgg aag agc 1392Lys Asn Pro Val Glu Leu Ile Glu Gly Trp Phe Ser Ser Trp Lys Ser 450 455 460 agc atc gct tca ttt ttt ttc atc atc ggc ctc atc atc ggg ctg ttt 1440Ser Ile Ala Ser Phe Phe Phe Ile Ile Gly Leu Ile Ile Gly Leu Phe 465 470 475 480 ctg gtg ctg cgc gtc ggc atc cac ctg tgc atc aag ctg aag cac acc 1488Leu Val Leu Arg Val Gly Ile His Leu Cys Ile Lys Leu Lys His Thr 485 490 495 aag aag cgc cag atc tat acc gac atc gag atg aat cgc ctg ggg aag 1536Lys Lys Arg Gln Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys 500 505 510 taa 153982512PRTArtificialSynthetic Construct 82Met Asn Phe Leu Leu Leu Thr Phe Ile Val Leu Pro Leu Cys Ser His 1 5 10 15 Ala Lys Phe Ser Ile Val Phe Pro Gln Ser Gln Lys Gly Asn Trp Lys 20 25 30 Asn Val Pro Ser Ser Tyr His Tyr Cys Pro Ser Ser Ser Asp Gln Asn 35 40 45 Trp His Asn Asp Leu Leu Gly Ile Thr Met Lys Val Lys Met Pro Lys 50 55 60 Thr His Lys Ala Ile Gln Ala Asp Gly Trp Met Cys His Ala Ala Lys 65 70 75 80 Trp Ile Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile Thr 85 90 95 His Ser Ile His Ser Ile Gln Pro Thr Ser Glu Gln Cys Lys Glu Ser 100 105 110 Ile Lys Gln Thr Lys Gln Gly Thr Trp Met Ser Pro Gly Phe Pro Pro 115 120 125 Gln Asn Cys Gly Tyr Ala Thr Val Thr Asp Ser Val Ala Val Val Val 130 135 140 Gln Ala Thr Pro His His Val Leu Val Asp Glu Tyr Thr Gly Glu Trp 145 150 155 160 Ile Asp Ser Gln Phe Pro Asn Gly Lys Cys Glu Thr Glu Glu Cys Glu 165 170 175 Thr Val His Asn Ser Thr Val Trp Tyr Ser Asp Tyr Lys Val Thr Gly 180 185 190 Leu Cys Asp Ala Thr Leu Val Asp Thr Glu Ile Thr Phe Phe Ser Glu 195 200 205 Asp Gly Lys Lys Glu Ser Ile Gly Lys Pro Asn Thr Gly Tyr Arg Ser 210 215 220 Asn Tyr Phe Ala Tyr Glu Lys Gly Asp Lys Val Cys Lys Met Asn Tyr 225 230 235 240 Cys Lys His Ala Gly Val Arg Leu Pro Ser Gly Val Trp Phe Glu Phe 245 250 255 Val Asp Gln Asp Val Tyr Ala Ala Ala Lys Leu Pro Glu Cys Pro Val 260 265 270 Gly Ala Thr Ile Ser Ala Pro Thr Gln Thr Ser Val Asp Val Ser Leu 275 280 285 Ile Leu Asp Val Glu Arg Ile Leu Asp Tyr Ser Leu Cys Gln Glu Thr 290 295 300 Trp Ser Lys Ile Arg Ser Lys Gln Pro Val Ser Pro Val Asp Leu Ser 305 310 315 320 Tyr Leu Ala Pro Lys Asn Pro Gly Thr Gly Pro Ala Phe Thr Ile Ile 325 330 335 Asn Gly Thr Leu Lys Tyr Phe Glu Thr Arg Tyr Ile Arg Ile Asp Ile 340 345 350 Asp Asn Pro Ile Ile Ser Lys Met Val Gly Lys Ile Ser Gly Ser Gln 355 360 365 Thr Glu Arg Glu Leu Trp Thr Glu Trp Phe Pro Tyr Glu Gly Val Glu 370 375 380 Ile Gly Pro Asn Gly Ile Leu Lys Thr Pro Thr Gly Tyr Lys Phe Pro 385 390 395 400 Leu Phe Met Ile Gly His Gly Met Leu Asp Ser Asp Leu His Lys Thr 405 410 415 Ser Gln Ala Glu Val Phe Glu His Pro His Leu Ala Glu Ala Pro Lys 420 425 430 Gln Leu Pro Glu Glu Glu Thr Leu Phe Phe Gly Asp Thr Gly Ile Ser 435 440 445 Lys Asn Pro Val Glu Leu Ile Glu Gly Trp Phe Ser Ser Trp Lys Ser 450 455 460 Ser Ile Ala Ser Phe Phe Phe Ile Ile Gly Leu Ile Ile Gly Leu Phe 465 470 475 480 Leu Val Leu Arg Val Gly Ile His Leu Cys Ile Lys Leu Lys His Thr 485 490 495 Lys Lys Arg Gln Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys 500 505 510 831518DNAArtificialFusion VSV-G Piry / Indiana 83atg acc gat aca gtg ctg ggc aag ttc cag atc gtg ttc ccc gac cag 48Met Thr Asp Thr Val Leu Gly Lys Phe Gln Ile Val Phe Pro Asp Gln 1 5 10 15 aac gag ctg gaa tgg acc ccc gtc gtg ggc gac agc cgg cat tgc cct 96Asn Glu Leu Glu Trp Thr Pro Val Val Gly Asp Ser Arg His Cys Pro 20 25 30 cag tcc agc gag atg cag ttc gac ggc agc aga agc cag acc atc ctg 144Gln Ser Ser Glu Met Gln Phe Asp Gly Ser Arg Ser Gln Thr Ile Leu 35 40 45 acc ggc aag gcc ccc gtg ggc atc aca ccc agc aag agc gac ggc ttc 192Thr Gly Lys Ala Pro Val Gly Ile Thr Pro Ser Lys Ser Asp Gly Phe 50 55 60 atc tgc cac gcc gcc aag tgg gtg acc acc tgc gac ttc cgg tgg tac 240Ile Cys His Ala Ala Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr 65 70 75 80 ggc ccc aag tac atc acc cac agc atc cac cac ctg cgg ccc acc acc 288Gly Pro Lys Tyr Ile Thr His Ser Ile His His Leu Arg Pro Thr Thr 85 90 95 tcc gac tgc gag aca gcc ctg cag cgg tac aag gac ggc agc ctg atc 336Ser Asp Cys Glu Thr Ala Leu Gln Arg Tyr Lys Asp Gly Ser Leu Ile 100 105 110 aac ctg ggc ttc cct ccc gag agc tgc ggc tac gcc acc gtg aca gac 384Asn Leu Gly Phe Pro Pro Glu Ser Cys Gly Tyr Ala Thr Val Thr Asp 115 120 125 agc gag gcc atg ctg gtg cag gtg acc ccc cac cac gtg ggc gtg gac 432Ser Glu Ala Met Leu Val Gln Val Thr Pro His His Val Gly Val Asp 130 135 140 gac tac cgg ggc cac tgg atc gac ccc ctg ttc cct ggc ggc gag tgc

480Asp Tyr Arg Gly His Trp Ile Asp Pro Leu Phe Pro Gly Gly Glu Cys 145 150 155 160 agc acc aat ttc tgc gat acc gtg cac aac agc agc gtg tgg att ccc 528Ser Thr Asn Phe Cys Asp Thr Val His Asn Ser Ser Val Trp Ile Pro 165 170 175 aag agc cag aaa acc gac atc tgc gcc cag agc ttc aag aac atc aag 576Lys Ser Gln Lys Thr Asp Ile Cys Ala Gln Ser Phe Lys Asn Ile Lys 180 185 190 atg acc gcc agc tac ccc agc gag gga gcc ctg gtg tcc gac cgg ttc 624Met Thr Ala Ser Tyr Pro Ser Glu Gly Ala Leu Val Ser Asp Arg Phe 195 200 205 gcc ttc cac agc gcc tac cac ccc aac atg ccc ggc agc acc gtg tgc 672Ala Phe His Ser Ala Tyr His Pro Asn Met Pro Gly Ser Thr Val Cys 210 215 220 atc atg gat ttc tgc gag cag aag ggc ctg cgg ttc acc aac ggc gag 720Ile Met Asp Phe Cys Glu Gln Lys Gly Leu Arg Phe Thr Asn Gly Glu 225 230 235 240 tgg atg ggc ctg aac gtg gag cag agc atc cgg gag aag aag atc agc 768Trp Met Gly Leu Asn Val Glu Gln Ser Ile Arg Glu Lys Lys Ile Ser 245 250 255 gcc atc ttc ccc aac tgc gtg gcc ggc acc gag atc cgg gcc acc ctg 816Ala Ile Phe Pro Asn Cys Val Ala Gly Thr Glu Ile Arg Ala Thr Leu 260 265 270 gaa tcc gag ggc gcc agg acc ctg acc tgg gag aca cag cgg atg ctg 864Glu Ser Glu Gly Ala Arg Thr Leu Thr Trp Glu Thr Gln Arg Met Leu 275 280 285 gac tac agc ctg tgc cag aac acc tgg gac aag gtg tcc cgg aaa gag 912Asp Tyr Ser Leu Cys Gln Asn Thr Trp Asp Lys Val Ser Arg Lys Glu 290 295 300 cct ctg tcc ccc ctg gac ctg agc tac ctg agc cct aga gcc cct ggc 960Pro Leu Ser Pro Leu Asp Leu Ser Tyr Leu Ser Pro Arg Ala Pro Gly 305 310 315 320 aag ggc atg gcc tac acc gtg atc aac ggc acc ctg cac agc gcc cac 1008Lys Gly Met Ala Tyr Thr Val Ile Asn Gly Thr Leu His Ser Ala His 325 330 335 gcc aag tat atc cgg acc tgg atc gac tac ggc gag atg aaa gag atc 1056Ala Lys Tyr Ile Arg Thr Trp Ile Asp Tyr Gly Glu Met Lys Glu Ile 340 345 350 aag ggc ggc agg ggc gag tac agc aag gcc cct gag ctg ctg tgg agc 1104Lys Gly Gly Arg Gly Glu Tyr Ser Lys Ala Pro Glu Leu Leu Trp Ser 355 360 365 cag tgg ttc gac ttc ggc ccc ttc aag atc ggc ccc aac ggc ctg ctg 1152Gln Trp Phe Asp Phe Gly Pro Phe Lys Ile Gly Pro Asn Gly Leu Leu 370 375 380 cac acc ggc aag acc ttc aag ttc cct ctg tat ctg atc gga gcc ggc 1200His Thr Gly Lys Thr Phe Lys Phe Pro Leu Tyr Leu Ile Gly Ala Gly 385 390 395 400 atc atc gac gag gac ctg cac gag ctg gac gaa gcc gcc cct atc gac 1248Ile Ile Asp Glu Asp Leu His Glu Leu Asp Glu Ala Ala Pro Ile Asp 405 410 415 cac ccc cag atg ccc gac gcc aag agc gtg ctg ccc gag gac gag gaa 1296His Pro Gln Met Pro Asp Ala Lys Ser Val Leu Pro Glu Asp Glu Glu 420 425 430 atc ttc ttc ggc gac acc ggc gtg agc aag aac ccc atc gag ctg atc 1344Ile Phe Phe Gly Asp Thr Gly Val Ser Lys Asn Pro Ile Glu Leu Ile 435 440 445 cag ggc tgg ttc agc aac tgg cgg agc agc atc gct tca ttt ttt ttc 1392Gln Gly Trp Phe Ser Asn Trp Arg Ser Ser Ile Ala Ser Phe Phe Phe 450 455 460 atc atc ggc ctc atc atc ggg ctg ttt ctg gtg ctg cgc gtc ggc atc 1440Ile Ile Gly Leu Ile Ile Gly Leu Phe Leu Val Leu Arg Val Gly Ile 465 470 475 480 cac ctg tgc atc aag ctg aag cac acc aag aag cgc cag atc tat acc 1488His Leu Cys Ile Lys Leu Lys His Thr Lys Lys Arg Gln Ile Tyr Thr 485 490 495 gac atc gag atg aat cgc ctg ggg aag taa 1518Asp Ile Glu Met Asn Arg Leu Gly Lys 500 505 84505PRTArtificialSynthetic Construct 84Met Thr Asp Thr Val Leu Gly Lys Phe Gln Ile Val Phe Pro Asp Gln 1 5 10 15 Asn Glu Leu Glu Trp Thr Pro Val Val Gly Asp Ser Arg His Cys Pro 20 25 30 Gln Ser Ser Glu Met Gln Phe Asp Gly Ser Arg Ser Gln Thr Ile Leu 35 40 45 Thr Gly Lys Ala Pro Val Gly Ile Thr Pro Ser Lys Ser Asp Gly Phe 50 55 60 Ile Cys His Ala Ala Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr 65 70 75 80 Gly Pro Lys Tyr Ile Thr His Ser Ile His His Leu Arg Pro Thr Thr 85 90 95 Ser Asp Cys Glu Thr Ala Leu Gln Arg Tyr Lys Asp Gly Ser Leu Ile 100 105 110 Asn Leu Gly Phe Pro Pro Glu Ser Cys Gly Tyr Ala Thr Val Thr Asp 115 120 125 Ser Glu Ala Met Leu Val Gln Val Thr Pro His His Val Gly Val Asp 130 135 140 Asp Tyr Arg Gly His Trp Ile Asp Pro Leu Phe Pro Gly Gly Glu Cys 145 150 155 160 Ser Thr Asn Phe Cys Asp Thr Val His Asn Ser Ser Val Trp Ile Pro 165 170 175 Lys Ser Gln Lys Thr Asp Ile Cys Ala Gln Ser Phe Lys Asn Ile Lys 180 185 190 Met Thr Ala Ser Tyr Pro Ser Glu Gly Ala Leu Val Ser Asp Arg Phe 195 200 205 Ala Phe His Ser Ala Tyr His Pro Asn Met Pro Gly Ser Thr Val Cys 210 215 220 Ile Met Asp Phe Cys Glu Gln Lys Gly Leu Arg Phe Thr Asn Gly Glu 225 230 235 240 Trp Met Gly Leu Asn Val Glu Gln Ser Ile Arg Glu Lys Lys Ile Ser 245 250 255 Ala Ile Phe Pro Asn Cys Val Ala Gly Thr Glu Ile Arg Ala Thr Leu 260 265 270 Glu Ser Glu Gly Ala Arg Thr Leu Thr Trp Glu Thr Gln Arg Met Leu 275 280 285 Asp Tyr Ser Leu Cys Gln Asn Thr Trp Asp Lys Val Ser Arg Lys Glu 290 295 300 Pro Leu Ser Pro Leu Asp Leu Ser Tyr Leu Ser Pro Arg Ala Pro Gly 305 310 315 320 Lys Gly Met Ala Tyr Thr Val Ile Asn Gly Thr Leu His Ser Ala His 325 330 335 Ala Lys Tyr Ile Arg Thr Trp Ile Asp Tyr Gly Glu Met Lys Glu Ile 340 345 350 Lys Gly Gly Arg Gly Glu Tyr Ser Lys Ala Pro Glu Leu Leu Trp Ser 355 360 365 Gln Trp Phe Asp Phe Gly Pro Phe Lys Ile Gly Pro Asn Gly Leu Leu 370 375 380 His Thr Gly Lys Thr Phe Lys Phe Pro Leu Tyr Leu Ile Gly Ala Gly 385 390 395 400 Ile Ile Asp Glu Asp Leu His Glu Leu Asp Glu Ala Ala Pro Ile Asp 405 410 415 His Pro Gln Met Pro Asp Ala Lys Ser Val Leu Pro Glu Asp Glu Glu 420 425 430 Ile Phe Phe Gly Asp Thr Gly Val Ser Lys Asn Pro Ile Glu Leu Ile 435 440 445 Gln Gly Trp Phe Ser Asn Trp Arg Ser Ser Ile Ala Ser Phe Phe Phe 450 455 460 Ile Ile Gly Leu Ile Ile Gly Leu Phe Leu Val Leu Arg Val Gly Ile 465 470 475 480 His Leu Cys Ile Lys Leu Lys His Thr Lys Lys Arg Gln Ile Tyr Thr 485 490 495 Asp Ile Glu Met Asn Arg Leu Gly Lys 500 505 851560DNAArtificialFusion VSV-G Isfahan / Indiana 85atg aca tcc gtg ctg ttt atg gtg ggc gtg ctg ctc gga gct ttc gga 48Met Thr Ser Val Leu Phe Met Val Gly Val Leu Leu Gly Ala Phe Gly 1 5 10 15 tct acc cac tgc agc atc cag atc gtg ttc ccc agc gag aca aag ctg 96Ser Thr His Cys Ser Ile Gln Ile Val Phe Pro Ser Glu Thr Lys Leu 20 25 30 gtg tgg aag ccc gtg ctg aag ggc acc cgg tac tgc ccc cag agc gcc 144Val Trp Lys Pro Val Leu Lys Gly Thr Arg Tyr Cys Pro Gln Ser Ala 35 40 45 gag ctg aac ctg gaa ccc gac ctg aaa acc atg gcc ttc gac agc aag 192Glu Leu Asn Leu Glu Pro Asp Leu Lys Thr Met Ala Phe Asp Ser Lys 50 55 60 gtg ccc atc ggc atc acc ccc agc aac agc gac ggc tac ctg tgc cac 240Val Pro Ile Gly Ile Thr Pro Ser Asn Ser Asp Gly Tyr Leu Cys His 65 70 75 80 gcc gcc aag tgg gtg acc acc tgc gac ttc cgg tgg tac ggc ccc aag 288Ala Ala Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys 85 90 95 tac atc acc cac agc gtg cac agc ctg cgg ccc acc gtg agc gac tgc 336Tyr Ile Thr His Ser Val His Ser Leu Arg Pro Thr Val Ser Asp Cys 100 105 110 aag gcc gcc gtg gaa gct tac aac gct ggc acc ctg atg tac ccc ggc 384Lys Ala Ala Val Glu Ala Tyr Asn Ala Gly Thr Leu Met Tyr Pro Gly 115 120 125 ttc ccc ccc gag agc tgc ggc tac gcc agc atc acc gac agc gag ttc 432Phe Pro Pro Glu Ser Cys Gly Tyr Ala Ser Ile Thr Asp Ser Glu Phe 130 135 140 tac gtg atg ctg gtg acc ccc cac ccc gtg gga gtg gac gac tac cgg 480Tyr Val Met Leu Val Thr Pro His Pro Val Gly Val Asp Asp Tyr Arg 145 150 155 160 ggc cac tgg gtg gac cct ctg ttc ccc acc tcc gag tgc aac agc aac 528Gly His Trp Val Asp Pro Leu Phe Pro Thr Ser Glu Cys Asn Ser Asn 165 170 175 ttc tgc gag aca gtg cac aac gcc acc atg tgg att ccc aag gat ctg 576Phe Cys Glu Thr Val His Asn Ala Thr Met Trp Ile Pro Lys Asp Leu 180 185 190 aaa acc cac gac gtg tgc agc cag gac ttc cag acc atc aga gtg agc 624Lys Thr His Asp Val Cys Ser Gln Asp Phe Gln Thr Ile Arg Val Ser 195 200 205 gtg atg tac cct cag acc aag ccc acc aag gga gct gac ctg aca ctg 672Val Met Tyr Pro Gln Thr Lys Pro Thr Lys Gly Ala Asp Leu Thr Leu 210 215 220 aag agc aag ttc cac gcc cac atg aag ggc gac aga gtg tgc aag atg 720Lys Ser Lys Phe His Ala His Met Lys Gly Asp Arg Val Cys Lys Met 225 230 235 240 aag ttc tgc aac aag aac ggc ctg cgg ctg ggc aac ggc gag tgg atc 768Lys Phe Cys Asn Lys Asn Gly Leu Arg Leu Gly Asn Gly Glu Trp Ile 245 250 255 gaa gtg ggc gac gag gtg atg ctg gac aac agc aag ctg ctg tcc ctg 816Glu Val Gly Asp Glu Val Met Leu Asp Asn Ser Lys Leu Leu Ser Leu 260 265 270 ttc ccc gac tgc ctg gtg ggc agc gtg gtg aag agc acc ctg ctg tcc 864Phe Pro Asp Cys Leu Val Gly Ser Val Val Lys Ser Thr Leu Leu Ser 275 280 285 gag ggc gtg cag acc gcc ctg tgg gag aca gac cgg ctg ctg gac tac 912Glu Gly Val Gln Thr Ala Leu Trp Glu Thr Asp Arg Leu Leu Asp Tyr 290 295 300 agc ctg tgc cag aac acc tgg gag aag atc gac cgg aaa gag ccc ctg 960Ser Leu Cys Gln Asn Thr Trp Glu Lys Ile Asp Arg Lys Glu Pro Leu 305 310 315 320 agc gcc gtc gac ctg agc tac ctg gcc cct aga agc ccc ggc aag ggc 1008Ser Ala Val Asp Leu Ser Tyr Leu Ala Pro Arg Ser Pro Gly Lys Gly 325 330 335 atg gcc tac atc gtg gcc aac ggc agc ctg atg agc gcc cct gcc cgg 1056Met Ala Tyr Ile Val Ala Asn Gly Ser Leu Met Ser Ala Pro Ala Arg 340 345 350 tac atc aga gtg tgg atc gac agc ccc atc ctg aaa gag atc aag ggc 1104Tyr Ile Arg Val Trp Ile Asp Ser Pro Ile Leu Lys Glu Ile Lys Gly 355 360 365 aag aaa gag agc gcc agc ggc atc gac acc gtg ctg tgg gag cag tgg 1152Lys Lys Glu Ser Ala Ser Gly Ile Asp Thr Val Leu Trp Glu Gln Trp 370 375 380 ctg ccc ttc aac ggc atg gaa ctg ggc ccc aac ggc ctg atc aag acc 1200Leu Pro Phe Asn Gly Met Glu Leu Gly Pro Asn Gly Leu Ile Lys Thr 385 390 395 400 aag agc ggc tac aag ttc ccc ctg tac ctg ctg ggc atg ggc atc gtg 1248Lys Ser Gly Tyr Lys Phe Pro Leu Tyr Leu Leu Gly Met Gly Ile Val 405 410 415 gac cag gac ctg cag gaa ctg agc agc gtc aac ccc gtg gac cac ccc 1296Asp Gln Asp Leu Gln Glu Leu Ser Ser Val Asn Pro Val Asp His Pro 420 425 430 cac gtg cct atc gcc cag gcc ttc gtg agc gag ggc gag gaa gtg ttc 1344His Val Pro Ile Ala Gln Ala Phe Val Ser Glu Gly Glu Glu Val Phe 435 440 445 ttc ggc gac acc ggc gtg agc aag aac ccc atc gag ctg atc agc ggc 1392Phe Gly Asp Thr Gly Val Ser Lys Asn Pro Ile Glu Leu Ile Ser Gly 450 455 460 tgg ttc agc gac tgg aaa agc agc atc gct tca ttt ttt ttc atc atc 1440Trp Phe Ser Asp Trp Lys Ser Ser Ile Ala Ser Phe Phe Phe Ile Ile 465 470 475 480 ggc ctc atc atc ggg ctg ttt ctg gtg ctg cgc gtc ggc atc cac ctg 1488Gly Leu Ile Ile Gly Leu Phe Leu Val Leu Arg Val Gly Ile His Leu 485 490 495 tgc atc aag ctg aag cac acc aag aag cgc cag atc tat acc gac atc 1536Cys Ile Lys Leu Lys His Thr Lys Lys Arg Gln Ile Tyr Thr Asp Ile 500 505 510 gag atg aat cgc ctg ggg aag taa 1560Glu Met Asn Arg Leu Gly Lys 515 86519PRTArtificialSynthetic Construct 86Met Thr Ser Val Leu Phe Met Val Gly Val Leu Leu Gly Ala Phe Gly 1 5 10 15 Ser Thr His Cys Ser Ile Gln Ile Val Phe Pro Ser Glu Thr Lys Leu 20 25 30 Val Trp Lys Pro Val Leu Lys Gly Thr Arg Tyr Cys Pro Gln Ser Ala 35 40 45 Glu Leu Asn Leu Glu Pro Asp Leu Lys Thr Met Ala Phe Asp Ser Lys 50 55 60 Val Pro Ile Gly Ile Thr Pro Ser Asn Ser Asp Gly Tyr Leu Cys His 65 70 75 80 Ala Ala Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys 85 90 95 Tyr Ile Thr His Ser Val His Ser Leu Arg Pro Thr Val Ser Asp Cys 100 105 110 Lys Ala Ala Val Glu Ala Tyr Asn Ala Gly Thr Leu Met Tyr Pro Gly 115 120 125 Phe Pro Pro Glu Ser Cys Gly Tyr Ala Ser Ile Thr Asp Ser Glu Phe 130 135 140 Tyr Val Met Leu Val Thr Pro His Pro Val Gly Val Asp Asp Tyr Arg 145 150 155 160 Gly His Trp Val Asp Pro Leu Phe Pro Thr Ser Glu Cys Asn Ser Asn 165 170 175 Phe Cys Glu Thr Val His Asn Ala Thr Met Trp Ile Pro Lys Asp Leu 180 185 190 Lys Thr His Asp Val Cys Ser Gln Asp Phe Gln Thr Ile Arg Val Ser 195 200 205 Val Met Tyr Pro Gln Thr Lys Pro Thr Lys Gly Ala Asp Leu Thr Leu 210 215 220 Lys Ser Lys Phe His Ala His Met Lys Gly Asp Arg Val Cys Lys Met 225 230 235 240 Lys Phe Cys Asn Lys Asn Gly Leu Arg Leu Gly Asn Gly Glu Trp Ile 245

250 255 Glu Val Gly Asp Glu Val Met Leu Asp Asn Ser Lys Leu Leu Ser Leu 260 265 270 Phe Pro Asp Cys Leu Val Gly Ser Val Val Lys Ser Thr Leu Leu Ser 275 280 285 Glu Gly Val Gln Thr Ala Leu Trp Glu Thr Asp Arg Leu Leu Asp Tyr 290 295 300 Ser Leu Cys Gln Asn Thr Trp Glu Lys Ile Asp Arg Lys Glu Pro Leu 305 310 315 320 Ser Ala Val Asp Leu Ser Tyr Leu Ala Pro Arg Ser Pro Gly Lys Gly 325 330 335 Met Ala Tyr Ile Val Ala Asn Gly Ser Leu Met Ser Ala Pro Ala Arg 340 345 350 Tyr Ile Arg Val Trp Ile Asp Ser Pro Ile Leu Lys Glu Ile Lys Gly 355 360 365 Lys Lys Glu Ser Ala Ser Gly Ile Asp Thr Val Leu Trp Glu Gln Trp 370 375 380 Leu Pro Phe Asn Gly Met Glu Leu Gly Pro Asn Gly Leu Ile Lys Thr 385 390 395 400 Lys Ser Gly Tyr Lys Phe Pro Leu Tyr Leu Leu Gly Met Gly Ile Val 405 410 415 Asp Gln Asp Leu Gln Glu Leu Ser Ser Val Asn Pro Val Asp His Pro 420 425 430 His Val Pro Ile Ala Gln Ala Phe Val Ser Glu Gly Glu Glu Val Phe 435 440 445 Phe Gly Asp Thr Gly Val Ser Lys Asn Pro Ile Glu Leu Ile Ser Gly 450 455 460 Trp Phe Ser Asp Trp Lys Ser Ser Ile Ala Ser Phe Phe Phe Ile Ile 465 470 475 480 Gly Leu Ile Ile Gly Leu Phe Leu Val Leu Arg Val Gly Ile His Leu 485 490 495 Cys Ile Lys Leu Lys His Thr Lys Lys Arg Gln Ile Tyr Thr Asp Ile 500 505 510 Glu Met Asn Arg Leu Gly Lys 515 871536DNAArtificialFusion VSV-G New Jersey / Indiana 87atg agc atc atc agc tat atc gcc ttt ctg ctg ctg atc gac agc acc 48Met Ser Ile Ile Ser Tyr Ile Ala Phe Leu Leu Leu Ile Asp Ser Thr 1 5 10 15 ctg ggc atc ccc atc ttc gtg ccc agc ggc cag aac atc agc tgg cag 96Leu Gly Ile Pro Ile Phe Val Pro Ser Gly Gln Asn Ile Ser Trp Gln 20 25 30 ccc gtg atc cag ccc ttc gac tac cag tgc ccc atc cac ggc aac ctg 144Pro Val Ile Gln Pro Phe Asp Tyr Gln Cys Pro Ile His Gly Asn Leu 35 40 45 ccc aac acc atg ggc ctg agc gcc acc aag ctg acc atc aag agc ccc 192Pro Asn Thr Met Gly Leu Ser Ala Thr Lys Leu Thr Ile Lys Ser Pro 50 55 60 agc gtg ttc agc acc gac aag gtg tcc ggc tgg atc tgc cac gcc gcc 240Ser Val Phe Ser Thr Asp Lys Val Ser Gly Trp Ile Cys His Ala Ala 65 70 75 80 gag tgg aaa acc acc tgc gac tac cgg tgg tac ggc ccc cag tac atc 288Glu Trp Lys Thr Thr Cys Asp Tyr Arg Trp Tyr Gly Pro Gln Tyr Ile 85 90 95 acc cac agc atc cac ccc atc agc ccc acc atc gac gag tgc aag cgg 336Thr His Ser Ile His Pro Ile Ser Pro Thr Ile Asp Glu Cys Lys Arg 100 105 110 atc atc agc cgg atc gcc agc ggc acc gac gag gac ctg ggc ttc cca 384Ile Ile Ser Arg Ile Ala Ser Gly Thr Asp Glu Asp Leu Gly Phe Pro 115 120 125 ccc cag agc tgc ggc tgg gcc agc gtg acc acc gtg agc aac acc aac 432Pro Gln Ser Cys Gly Trp Ala Ser Val Thr Thr Val Ser Asn Thr Asn 130 135 140 tac aag gtg gtg ccc cac agc gtg cac ctg gaa ccc tac ggc ggc cac 480Tyr Lys Val Val Pro His Ser Val His Leu Glu Pro Tyr Gly Gly His 145 150 155 160 tgg atc gac cac gac ttc aac ggc ggc gag tgc cgg gag aaa gtg tgc 528Trp Ile Asp His Asp Phe Asn Gly Gly Glu Cys Arg Glu Lys Val Cys 165 170 175 gag atg aag ggc aac cac agc atc tgg atc acc gac gag aca gtg cag 576Glu Met Lys Gly Asn His Ser Ile Trp Ile Thr Asp Glu Thr Val Gln 180 185 190 cac gag tgc gag aag cac atc gag gaa gtg gag ggc atc atg tac ggc 624His Glu Cys Glu Lys His Ile Glu Glu Val Glu Gly Ile Met Tyr Gly 195 200 205 aac gcc ccc agg ggc gac gcc atc tac atc aac aac ttc atc atc gac 672Asn Ala Pro Arg Gly Asp Ala Ile Tyr Ile Asn Asn Phe Ile Ile Asp 210 215 220 aag cac cac cgg gtg tac cgg ttc ggc ggc tcc tgc cgg atg aag ttc 720Lys His His Arg Val Tyr Arg Phe Gly Gly Ser Cys Arg Met Lys Phe 225 230 235 240 tgc aac aag gac ggc atc aag ttc acc aga ggc gac tgg gtg gag aaa 768Cys Asn Lys Asp Gly Ile Lys Phe Thr Arg Gly Asp Trp Val Glu Lys 245 250 255 acc gcc ggc acc ctg acc aac atc tac gag aac atc ccc gag tgc gcc 816Thr Ala Gly Thr Leu Thr Asn Ile Tyr Glu Asn Ile Pro Glu Cys Ala 260 265 270 gac ggc aca ctg gtg tcc ggc cac aga ccc ggc ctg gac ctg atc gac 864Asp Gly Thr Leu Val Ser Gly His Arg Pro Gly Leu Asp Leu Ile Asp 275 280 285 acc gtg ttc aac ctg gaa aac gtg gtg gag tac acc ctg tgc gag ggc 912Thr Val Phe Asn Leu Glu Asn Val Val Glu Tyr Thr Leu Cys Glu Gly 290 295 300 acc aag cgg aag atc aac aag cag gaa aag ctg acc agc gtc gac ctg 960Thr Lys Arg Lys Ile Asn Lys Gln Glu Lys Leu Thr Ser Val Asp Leu 305 310 315 320 agc tac ctg gcc ccc agg atc ggc ggc ttc ggc agc gtg ttc cgc gtg 1008Ser Tyr Leu Ala Pro Arg Ile Gly Gly Phe Gly Ser Val Phe Arg Val 325 330 335 cgg aat ggg acc ctg gaa aga gga agc aca aca tac att cgg atc gaa 1056Arg Asn Gly Thr Leu Glu Arg Gly Ser Thr Thr Tyr Ile Arg Ile Glu 340 345 350 gtg gaa ggc ccc gtg gtg gac agc ctg aac ggc atc gac ccc cgg acc 1104Val Glu Gly Pro Val Val Asp Ser Leu Asn Gly Ile Asp Pro Arg Thr 355 360 365 aac gcc agc cgg gtg ttc tgg gac gac tgg gag ctg gac ggc aac atc 1152Asn Ala Ser Arg Val Phe Trp Asp Asp Trp Glu Leu Asp Gly Asn Ile 370 375 380 tac cag ggc ttc aat ggc gtg tac aag ggc aag gat ggc aag atc cac 1200Tyr Gln Gly Phe Asn Gly Val Tyr Lys Gly Lys Asp Gly Lys Ile His 385 390 395 400 atc ccc ctg aac atg atc gag agc ggc atc atc gac gac gag ctg cag 1248Ile Pro Leu Asn Met Ile Glu Ser Gly Ile Ile Asp Asp Glu Leu Gln 405 410 415 cac gcc ttc cag gcc gac atc atc ccc cac ccc cac tac gac gac gac 1296His Ala Phe Gln Ala Asp Ile Ile Pro His Pro His Tyr Asp Asp Asp 420 425 430 gag atc cgg gag gac gac atc ttc ttc gac aac acc ggc gag aac ggc 1344Glu Ile Arg Glu Asp Asp Ile Phe Phe Asp Asn Thr Gly Glu Asn Gly 435 440 445 aac ccc gtg gac gcc gtg gtg gaa tgg gtg tcc gga tgg ggc agc agc 1392Asn Pro Val Asp Ala Val Val Glu Trp Val Ser Gly Trp Gly Ser Ser 450 455 460 atc gct tca ttt ttt ttc atc atc ggc ctc atc atc ggg ctg ttt ctg 1440Ile Ala Ser Phe Phe Phe Ile Ile Gly Leu Ile Ile Gly Leu Phe Leu 465 470 475 480 gtg ctg cgc gtc ggc atc cac ctg tgc atc aag ctg aag cac acc aag 1488Val Leu Arg Val Gly Ile His Leu Cys Ile Lys Leu Lys His Thr Lys 485 490 495 aag cgc cag atc tat acc gac atc gag atg aat cgc ctg ggg aag taa 1536Lys Arg Gln Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys 500 505 510 88511PRTArtificialSynthetic Construct 88Met Ser Ile Ile Ser Tyr Ile Ala Phe Leu Leu Leu Ile Asp Ser Thr 1 5 10 15 Leu Gly Ile Pro Ile Phe Val Pro Ser Gly Gln Asn Ile Ser Trp Gln 20 25 30 Pro Val Ile Gln Pro Phe Asp Tyr Gln Cys Pro Ile His Gly Asn Leu 35 40 45 Pro Asn Thr Met Gly Leu Ser Ala Thr Lys Leu Thr Ile Lys Ser Pro 50 55 60 Ser Val Phe Ser Thr Asp Lys Val Ser Gly Trp Ile Cys His Ala Ala 65 70 75 80 Glu Trp Lys Thr Thr Cys Asp Tyr Arg Trp Tyr Gly Pro Gln Tyr Ile 85 90 95 Thr His Ser Ile His Pro Ile Ser Pro Thr Ile Asp Glu Cys Lys Arg 100 105 110 Ile Ile Ser Arg Ile Ala Ser Gly Thr Asp Glu Asp Leu Gly Phe Pro 115 120 125 Pro Gln Ser Cys Gly Trp Ala Ser Val Thr Thr Val Ser Asn Thr Asn 130 135 140 Tyr Lys Val Val Pro His Ser Val His Leu Glu Pro Tyr Gly Gly His 145 150 155 160 Trp Ile Asp His Asp Phe Asn Gly Gly Glu Cys Arg Glu Lys Val Cys 165 170 175 Glu Met Lys Gly Asn His Ser Ile Trp Ile Thr Asp Glu Thr Val Gln 180 185 190 His Glu Cys Glu Lys His Ile Glu Glu Val Glu Gly Ile Met Tyr Gly 195 200 205 Asn Ala Pro Arg Gly Asp Ala Ile Tyr Ile Asn Asn Phe Ile Ile Asp 210 215 220 Lys His His Arg Val Tyr Arg Phe Gly Gly Ser Cys Arg Met Lys Phe 225 230 235 240 Cys Asn Lys Asp Gly Ile Lys Phe Thr Arg Gly Asp Trp Val Glu Lys 245 250 255 Thr Ala Gly Thr Leu Thr Asn Ile Tyr Glu Asn Ile Pro Glu Cys Ala 260 265 270 Asp Gly Thr Leu Val Ser Gly His Arg Pro Gly Leu Asp Leu Ile Asp 275 280 285 Thr Val Phe Asn Leu Glu Asn Val Val Glu Tyr Thr Leu Cys Glu Gly 290 295 300 Thr Lys Arg Lys Ile Asn Lys Gln Glu Lys Leu Thr Ser Val Asp Leu 305 310 315 320 Ser Tyr Leu Ala Pro Arg Ile Gly Gly Phe Gly Ser Val Phe Arg Val 325 330 335 Arg Asn Gly Thr Leu Glu Arg Gly Ser Thr Thr Tyr Ile Arg Ile Glu 340 345 350 Val Glu Gly Pro Val Val Asp Ser Leu Asn Gly Ile Asp Pro Arg Thr 355 360 365 Asn Ala Ser Arg Val Phe Trp Asp Asp Trp Glu Leu Asp Gly Asn Ile 370 375 380 Tyr Gln Gly Phe Asn Gly Val Tyr Lys Gly Lys Asp Gly Lys Ile His 385 390 395 400 Ile Pro Leu Asn Met Ile Glu Ser Gly Ile Ile Asp Asp Glu Leu Gln 405 410 415 His Ala Phe Gln Ala Asp Ile Ile Pro His Pro His Tyr Asp Asp Asp 420 425 430 Glu Ile Arg Glu Asp Asp Ile Phe Phe Asp Asn Thr Gly Glu Asn Gly 435 440 445 Asn Pro Val Asp Ala Val Val Glu Trp Val Ser Gly Trp Gly Ser Ser 450 455 460 Ile Ala Ser Phe Phe Phe Ile Ile Gly Leu Ile Ile Gly Leu Phe Leu 465 470 475 480 Val Leu Arg Val Gly Ile His Leu Cys Ile Lys Leu Lys His Thr Lys 485 490 495 Lys Arg Gln Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys 500 505 510 891557DNAArtificialFusion VSV-G New Jersey / Indiana 89atg ctg tca tat ctg atc ttt gcc ctg gct gtg agc cca atc ctc gga 48Met Leu Ser Tyr Leu Ile Phe Ala Leu Ala Val Ser Pro Ile Leu Gly 1 5 10 15 aag atc gaa atc gtg ttc cca caa cac acc aca ggg gac tgg aag cgc 96Lys Ile Glu Ile Val Phe Pro Gln His Thr Thr Gly Asp Trp Lys Arg 20 25 30 gtg ccc cac gag tac aac tac tgc ccg acc tca gcc gac aag aat agc 144Val Pro His Glu Tyr Asn Tyr Cys Pro Thr Ser Ala Asp Lys Asn Ser 35 40 45 cac ggc acg cag acc ggc atc cct gtg gag ctg acc atg ccc aag ggg 192His Gly Thr Gln Thr Gly Ile Pro Val Glu Leu Thr Met Pro Lys Gly 50 55 60 ctc aca acg cac caa gtc gaa ggc ttc atg tgc cac agc gct ctc tgg 240Leu Thr Thr His Gln Val Glu Gly Phe Met Cys His Ser Ala Leu Trp 65 70 75 80 atg aca acc tgc gat ttt cgc tgg tat ggc ccc aag tac atc acg cac 288Met Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile Thr His 85 90 95 agc atc cac aat gag gaa cca acc gac tac cag tgc ctc gaa gcc atc 336Ser Ile His Asn Glu Glu Pro Thr Asp Tyr Gln Cys Leu Glu Ala Ile 100 105 110 aag tca tac aag gat ggg gtg agc ttc aac ccc ggc ttc ccg ccc caa 384Lys Ser Tyr Lys Asp Gly Val Ser Phe Asn Pro Gly Phe Pro Pro Gln 115 120 125 tca tgt ggc tac ggc acc gtg acc gac gcc gag gcc cac atc gtg acc 432Ser Cys Gly Tyr Gly Thr Val Thr Asp Ala Glu Ala His Ile Val Thr 130 135 140 gtg aca ccc cac tca gtc aag gtg gac gag tac aca ggc gaa tgg atc 480Val Thr Pro His Ser Val Lys Val Asp Glu Tyr Thr Gly Glu Trp Ile 145 150 155 160 gac ccc cac ttc atc ggg ggc cgc tgt aag ggc caa atc tgc gag acc 528Asp Pro His Phe Ile Gly Gly Arg Cys Lys Gly Gln Ile Cys Glu Thr 165 170 175 gtg cac aac agc acc aag tgg ttt acg tca tca gac ggc gaa agc gtg 576Val His Asn Ser Thr Lys Trp Phe Thr Ser Ser Asp Gly Glu Ser Val 180 185 190 tgc agc caa ctg ttt acg ctc gtg ggc ggc atc ttc ttt agc gac agc 624Cys Ser Gln Leu Phe Thr Leu Val Gly Gly Ile Phe Phe Ser Asp Ser 195 200 205 gag gag atc acc agc atg ggc ctc ccg gag aca gga atc cgc agc aac 672Glu Glu Ile Thr Ser Met Gly Leu Pro Glu Thr Gly Ile Arg Ser Asn 210 215 220 tac ttt ccg tac atc agc acc gag gga atc tgt aag atg cct ttt tgc 720Tyr Phe Pro Tyr Ile Ser Thr Glu Gly Ile Cys Lys Met Pro Phe Cys 225 230 235 240 cgc aag cag gga tat aag ctg aag aat gac ctg tgg ttc cag atc atg 768Arg Lys Gln Gly Tyr Lys Leu Lys Asn Asp Leu Trp Phe Gln Ile Met 245 250 255 gac ccg gac ctg gac aag acc gtc cgc gat ctg ccc cac atc aag gac 816Asp Pro Asp Leu Asp Lys Thr Val Arg Asp Leu Pro His Ile Lys Asp 260 265 270 tgt gat ctg tca tca agc atc atc acc ccc gga gaa cac gcc acg gac 864Cys Asp Leu Ser Ser Ser Ile Ile Thr Pro Gly Glu His Ala Thr Asp 275 280 285 atc agc ctc atc agc gat gtg gag cgc atc ctc gac tac gct ctc tgc 912Ile Ser Leu Ile Ser Asp Val Glu Arg Ile Leu Asp Tyr Ala Leu Cys 290 295 300 cag aac aca tgg agc aag atc gaa agc ggc gaa ccc atc acc cca gtg 960Gln Asn Thr Trp Ser Lys Ile Glu Ser Gly Glu Pro Ile Thr Pro Val 305 310 315 320 gac ctg agc tat ctc ggc cca aag aac ccc ggc gtg ggg ccc gtg ttc 1008Asp Leu Ser Tyr Leu Gly Pro Lys Asn Pro Gly Val Gly Pro Val Phe 325 330 335 acc atc atc aac ggg agc ctg cac tac ttt aca agc aag tat ctg cgc 1056Thr Ile Ile Asn Gly Ser Leu His Tyr Phe Thr Ser Lys Tyr Leu Arg 340 345 350 gtg gag ctc gaa agc cca gtc atc ccc cgc atg gag ggg aag gtg gcc 1104Val Glu Leu Glu Ser Pro Val Ile Pro Arg Met Glu Gly Lys Val Ala 355 360 365 ggg acc cgc atc gtg cgc cag ctg tgg gac cag tgg ttc cct ttt ggc

1152Gly Thr Arg Ile Val Arg Gln Leu Trp Asp Gln Trp Phe Pro Phe Gly 370 375 380 gag gtg gaa atc ggc ccc aac ggc gtg ctg aag acc aag caa gga tat 1200Glu Val Glu Ile Gly Pro Asn Gly Val Leu Lys Thr Lys Gln Gly Tyr 385 390 395 400 aag ttc ccg ctg cac atc atc ggg acg ggc gaa gtg gac agc gat atc 1248Lys Phe Pro Leu His Ile Ile Gly Thr Gly Glu Val Asp Ser Asp Ile 405 410 415 aag atg gag cgc gtg gtc aag cac tgg gag cac cca cac atc gag gct 1296Lys Met Glu Arg Val Val Lys His Trp Glu His Pro His Ile Glu Ala 420 425 430 gct cag acc ttt ctc aag aag gac gat acc ggc gaa gtc ctg tat tac 1344Ala Gln Thr Phe Leu Lys Lys Asp Asp Thr Gly Glu Val Leu Tyr Tyr 435 440 445 ggg gat acg gga gtg agc aag aac cct gtg gag ctg gtg gaa ggc tgg 1392Gly Asp Thr Gly Val Ser Lys Asn Pro Val Glu Leu Val Glu Gly Trp 450 455 460 ttc agc gga tgg cgc agc agc atc gct tca ttt ttt ttc atc atc ggc 1440Phe Ser Gly Trp Arg Ser Ser Ile Ala Ser Phe Phe Phe Ile Ile Gly 465 470 475 480 ctc atc atc ggg ctg ttt ctg gtg ctg cgc gtc ggc atc cac ctg tgc 1488Leu Ile Ile Gly Leu Phe Leu Val Leu Arg Val Gly Ile His Leu Cys 485 490 495 atc aag ctg aag cac acc aag aag cgc cag atc tat acc gac atc gag 1536Ile Lys Leu Lys His Thr Lys Lys Arg Gln Ile Tyr Thr Asp Ile Glu 500 505 510 atg aat cgc ctg ggg aag taa 1557Met Asn Arg Leu Gly Lys 515 90518PRTArtificialSynthetic Construct 90Met Leu Ser Tyr Leu Ile Phe Ala Leu Ala Val Ser Pro Ile Leu Gly 1 5 10 15 Lys Ile Glu Ile Val Phe Pro Gln His Thr Thr Gly Asp Trp Lys Arg 20 25 30 Val Pro His Glu Tyr Asn Tyr Cys Pro Thr Ser Ala Asp Lys Asn Ser 35 40 45 His Gly Thr Gln Thr Gly Ile Pro Val Glu Leu Thr Met Pro Lys Gly 50 55 60 Leu Thr Thr His Gln Val Glu Gly Phe Met Cys His Ser Ala Leu Trp 65 70 75 80 Met Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile Thr His 85 90 95 Ser Ile His Asn Glu Glu Pro Thr Asp Tyr Gln Cys Leu Glu Ala Ile 100 105 110 Lys Ser Tyr Lys Asp Gly Val Ser Phe Asn Pro Gly Phe Pro Pro Gln 115 120 125 Ser Cys Gly Tyr Gly Thr Val Thr Asp Ala Glu Ala His Ile Val Thr 130 135 140 Val Thr Pro His Ser Val Lys Val Asp Glu Tyr Thr Gly Glu Trp Ile 145 150 155 160 Asp Pro His Phe Ile Gly Gly Arg Cys Lys Gly Gln Ile Cys Glu Thr 165 170 175 Val His Asn Ser Thr Lys Trp Phe Thr Ser Ser Asp Gly Glu Ser Val 180 185 190 Cys Ser Gln Leu Phe Thr Leu Val Gly Gly Ile Phe Phe Ser Asp Ser 195 200 205 Glu Glu Ile Thr Ser Met Gly Leu Pro Glu Thr Gly Ile Arg Ser Asn 210 215 220 Tyr Phe Pro Tyr Ile Ser Thr Glu Gly Ile Cys Lys Met Pro Phe Cys 225 230 235 240 Arg Lys Gln Gly Tyr Lys Leu Lys Asn Asp Leu Trp Phe Gln Ile Met 245 250 255 Asp Pro Asp Leu Asp Lys Thr Val Arg Asp Leu Pro His Ile Lys Asp 260 265 270 Cys Asp Leu Ser Ser Ser Ile Ile Thr Pro Gly Glu His Ala Thr Asp 275 280 285 Ile Ser Leu Ile Ser Asp Val Glu Arg Ile Leu Asp Tyr Ala Leu Cys 290 295 300 Gln Asn Thr Trp Ser Lys Ile Glu Ser Gly Glu Pro Ile Thr Pro Val 305 310 315 320 Asp Leu Ser Tyr Leu Gly Pro Lys Asn Pro Gly Val Gly Pro Val Phe 325 330 335 Thr Ile Ile Asn Gly Ser Leu His Tyr Phe Thr Ser Lys Tyr Leu Arg 340 345 350 Val Glu Leu Glu Ser Pro Val Ile Pro Arg Met Glu Gly Lys Val Ala 355 360 365 Gly Thr Arg Ile Val Arg Gln Leu Trp Asp Gln Trp Phe Pro Phe Gly 370 375 380 Glu Val Glu Ile Gly Pro Asn Gly Val Leu Lys Thr Lys Gln Gly Tyr 385 390 395 400 Lys Phe Pro Leu His Ile Ile Gly Thr Gly Glu Val Asp Ser Asp Ile 405 410 415 Lys Met Glu Arg Val Val Lys His Trp Glu His Pro His Ile Glu Ala 420 425 430 Ala Gln Thr Phe Leu Lys Lys Asp Asp Thr Gly Glu Val Leu Tyr Tyr 435 440 445 Gly Asp Thr Gly Val Ser Lys Asn Pro Val Glu Leu Val Glu Gly Trp 450 455 460 Phe Ser Gly Trp Arg Ser Ser Ile Ala Ser Phe Phe Phe Ile Ile Gly 465 470 475 480 Leu Ile Ile Gly Leu Phe Leu Val Leu Arg Val Gly Ile His Leu Cys 485 490 495 Ile Lys Leu Lys His Thr Lys Lys Arg Gln Ile Tyr Thr Asp Ile Glu 500 505 510 Met Asn Arg Leu Gly Lys 515

Claims

1-22. (canceled)

23. A lentiviral vector particle (i) which is pseudotyped with at least one determined heterologous viral envelope protein originating from a RNA virus and (ii) which comprises in its genome at least one recombinant polynucleotide encoding at least one polypeptide comprising at least one epitope of a pre-erythrocytic stage antigen of a Plasmodium parasite capable of infecting a mammalian host.

24. The lentiviral vector particle according to claim 23, which is a replication-incompetent HIV-based vector particle.

25. The lentiviral vector particle according to claim 23, wherein the at least one recombinant polynucleotide comprises a nucleic acid sequence encoding a polypeptide(s) of an antigen from the circumsporozoite protein (CSP) of a Plasmodium parasite infecting humans, or encoding a polypeptide of an antigen selected from the sporozoite surface protein 2 (TRAP/SSP2), liver-stage antigen (LSA), LSA3, Pf exported protein 1 (Pf Exp1), Pf antigen 2 sporozoite and liver stage antigen (SALSA), sporozoite threonine and asparagines-rich (STARP).

26. The lentiviral vector particle according to claim 23, wherein the at least one recombinant polynucleotide encodes a polypeptide chosen from merozoite surface protein, Merozoite surface protein 1 (MSP-1), Merozoite surface protein 2 (MSP-2), Merozoite surface protein 3 (MSP-3), Merozoite surface protein 4 (MSP-4), Merozoite surface protein 6 (MSP-6), MSP3-GLURP fusion protein, Ring-infected erythrocyte surface antigen (RESA), Rhoptry associated protein 1 (RAP-1), Apical membrane antigen 1 (AMA-1), Erythrocyte binding antigen (EBA-175), Erythrocyte membrane-associated giant protein or Antigen 332 (Ag332), dnaK-type molecular chaperone, Glutamate-rich protein (GLURP); MSP3-GLURP fusion protein, Erythrocyte membrane protein 1 (EMP-1), Serine repeat antigen (SERA), Clustered-asparagine-rich protein (CARP), Cirumsporozoite protein-related antigen precursor (CRA), Cytoadherence-linked asexual protein (CLAG), Acid basic repeat antigen (ABRA), 101 kDa malaria antigen, Rhoptry antigen protein (RAP-2), Knob-associated histidine-rich protein (KHRP), Rhoptry antigen protein (RAP), Cysteine protease, Hypothetical protein PFE1325w, Protective antigen (MAg-1), Fructose-bisphosphate aldolase, Ribosomal phosphoprotein P0, P-type ATPase, Glucose-regulated protein (GRP78), Asparagine and aspartate-rich protein (AARP1), Interspersed repeat antigen or PFE0070w, Sexual stage and sporozoite surface antigen, Antigen Pfg27/25, Antigen QF122, 11-1 polypeptide, Gametocyte-specific surface protein (Pfs230), Ookinete surface protein (P25), Chitinase, and Multidrug resistance protein (MRP).

27. The lentiviral vector particle according to claim 23, wherein the at least one recombinant polynucleotide has a mammalian codon optimized nucleotide sequence.

28. The lentiviral vector particle according to claim 23, wherein a the at least one recombinant polynucleotide encodes at least a polypeptide of the CSP antigen, said polypeptide being devoid of the GPI-anchoring motif of said CSP.

29. The lentiviral vector particle according to claim 23, which is an integration-deficient vector particle.

30. The lentiviral vector particle according to claim 23, which is an integration-competent vector particle.

31. The lentiviral vector particle according to claim 23, which is pseudotyped with at least one viral transmembrane glycosylated (G) envelope protein(s) of a Vesicular Stomatitis Virus (VSV) chosen from VSV-G protein(s) of the Indiana strain, VSV-G protein(s) of the New Jersey strain, VSV-G protein(s) of the Cocal strain, VSV-G protein of the Isfahan strain, VSV-G protein(s) of Chandipura strain, VSV-G protein(s) of Pyri strain and VSV-G protein(s) of the SVCV strain.

32. The lentiviral vector particle according to claim 23, recovered from mammalian cells co-transfected with: a) a vector plasmid comprising lentiviral cis-active sequences necessary for packaging, reverse transcription, and transcription; a functional lentiviral DNA flap element; and a polynucleotide of a truncated mammalian codon-optimized sequence of the cs gene of a Plasmodium parasite, under the control of regulatory expression sequences; b) a VSV-G envelope expression plasmid comprising a polynucleotide encoding a VSV-G envelope protein or envelope proteins under the control of regulatory expression sequences; and c) an encapsidation plasmid comprising lentiviral gag-pol coding sequences suitable for the production of integration-competent vector particles or modified gag-pol coding sequences suitable for the production of integration-deficient vector particles, wherein said gag-pol sequences are from the same lentivirus sub-family as the DNA flap element, wherein said gag-pol or modified gag-pol sequences are under the control of regulatory expression sequences.

33. The lentiviral vector particle according to claim 23, recovered from a stable cell line transfected with a vector plasmid comprising (i) lentiviral, especially HIV-1, cis-active sequences necessary for packaging, reverse transcription, and transcription and further comprising a functional lentiviral, especially HIV-1, DNA flap element and optionally comprising cis-active sequences necessary for integration, said vector plasmid further comprising (ii) a polynucleotide of a truncated mammalian, especially human, codon-optimized sequence of the cs gene of a Plasmodium parasite, under the control of regulatory expression sequences, especially a promoter; a VSV-G envelope expression plasmid comprising a polynucleotide encoding a VSV-G envelope protein or envelope proteins, wherein said polynucleotide is under the control of regulating expression sequences, in particular regulatory expression sequences comprising an inducible promoter, and; an encapsidation plasmid, wherein the encapsidation plasmid either comprises lentiviral, especially HIV-1, gag-pol coding sequences suitable for the production of integration-competent vector particles or modified gag-pol coding sequences suitable for the production of integration-deficient vector particles, wherein said gag-pol sequences are from the same lentivirus sub-family as the DNA flap element, wherein said lentiviral gag-pol or modified gag-pol sequence is under the control of regulating expression sequences.

34. The lentiviral vector particle according to claim 23, which comprises in its genome lentiviral-based sequences devoid of functional lentiviral genes, wherein said lentiviral-based sequences comprise: cis-active sequences necessary for packaging, reverse transcription, and transcription and a functional lentiviral DNA flap element, wherein said cis-acting sequences comprise at least one modification selected from: a) the 3'LTR sequence from the lentiviral genome is truncated and devoid of the enhancer of the U3 region; b) the 3' LTR sequence from the lentiviral genome is truncated and devoid of the U3 region or partly deleted in the U3 region; and c) the U3 region of the LTR5' is replaced by a non lentiviral U3 region or by a promoter suitable to drive tat-independent primary transcription.

35. A combination of lentiviral vector particles for separate administration to a mammalian host, which comprises: (i) a lentiviral vector particle according to claim 23, pseudotyped with at least one first determined heterologous viral envelope protein; and (ii) provided separately from the lentiviral vector particle in (i), a lentiviral vector particle according to claim 23, pseudotyped with at least one second determined heterologous viral envelope protein; wherein said first and second at least one viral envelope protein do not sero-neutralize with each other and are suitable for in vivo transduction of mammalian cells.

36. A combination of lentiviral vector particles according to claim 35, wherein said first and second at least one viral envelope proteins are selected from: VSV-G of Indiana strain and VSV-G of New Jersey strain; modified versions of native VSV-G of Indiana strain and VSV-G of New Jersey strain; a chimeric VSV-G protein wherein at least one of the following domains is from an Indiana strain: the export determinant YTDIE, the cytoplasmic tail, the transmembrane domain and the cytoplasmic domain; and the first at least one viral envelope protein is either VSV-G of Indiana strain or VSV-G of New Jersey strain and the second at least one viral envelope protein is selected from VSV-G protein(s) of the Cocal strain, VSV-G protein of the Isfahan strain, VSV-G protein(s) of Chandipura strain, VSV-G protein(s) of Pyri strain and VSV-G protein(s) of the SVCV strain.

37. A combination of separately provided lentiviral vector particles according to claim 35, wherein the lentiviral particles encode distinct polypeptides including (i) a polypeptide of the CSP antigen or a polypeptide of the CSP antigen devoid of the GPI-anchoring motif and (ii) at least one polypeptide of an antigen of the malaria parasite chosen from sporozoite surface protein 2 (TRAP/SSP2), liver-stage antigen (LSA), Pf exported protein 1 (Pf Exp1), and Pf antigen 2, said distinct polypeptides of said antigens being either expressed from the same lentiviral particles or from distinct lentiviral particles.

38. A combination of separately provided lentiviral vector particles according to claim 35, wherein the lentiviral particles encode a polypeptide chosen from a merozoite surface protein 1 (Msp-1), merozoite surface protein 2 (Msp-2), apical membrane antigen 1 (AMA-1), serine repeat antigen (SERA), GLURP antigen, Pf 155/RESA (Ring infected erythrocyte surface antigen) or a RHOPTRY-associated protein 1 (RAP-1), and RHOPTRY-associated protein 2 (RAP-2).

39. A composition comprising a lentiviral vector particle according to claim 23 and a physiologically acceptable vehicle.

40. The composition of claim 39, further comprising an adjuvant and/or an immunostimulant.

41. A method of prophylactic immunization against malaria parasite infection or against parasite-induced pathology in a mammalian host, comprising administering an effective amount of a first lentiviral particle according to claim 23 to prime the cellular immune response of the host and later in time administering an effective amount of a second lentiviral particle according to claim 23 to boost the cellular immune response of the host, wherein the first and second lentiviral particles administered in each of the priming or boosting steps are pseudotyped with distinct envelope protein(s) which do not sero-neutralize with each other, and wherein said priming and boosting steps are separated in time by at least 6 weeks.

42. A method of prophylactic immunization against malaria parasite infection or against parasite-induced pathology in a mammalian host, comprising administering a lentiviral particle according to claim 23 in a dosage regimen comprising separately provided doses of said lentiviral particle, wherein a dose administered for priming the cellular immune response is a moderate dose and a dose administered for boosting the cellular immune response is higher than the dose for priming.

43. A method of prophylactic immunization against malaria parasite infection or against parasite-induced pathology in a mammalian host, comprising administering a lentiviral particle according to claim 23 in a dosage regimen comprising separately provided doses of said lentiviral particle according to claim 23, wherein the dose administered for priming and the dose administered for boosting the cellular immune response comprise from 10.sup.7 to 10.sup.9 lentiviral particles when integrative-competent vector particles are used and the dose administered for priming and for boosting the cellular immune response comprise from 10.sup.8 to 10.sup.10 lentiviral particles when integrative-incompetent vector particles are used.

44. The method of claim 43, wherein administering the lentiviral particle in the dosage regimen results in at least one of the following effects in the host: (I) eliciting sterile protection against malaria parasite infection, in a human host; (II) inhibiting extracellular forms of malaria parasite; (III) preventing hepatocytes infection by malaria parasite or inhibition of liver stage amplification of infection; (IV) eliciting a specific T-cell immune response against malaria parasite antigen(s); (V) eliciting a B-cell response against parasite antigen(s); (VI) controlling parasitemia so as to reduce or alleviate the effects of infection by the malaria parasite; (VII) eliciting a protective cellular immunity against the infection by the parasite or against the parasite-induced pathology; (VIII) eliciting memory T-cell immune response (IX) eliciting earlier and higher rebound of the CD4+ and CD8+ T-cell response during infection by the malaria parasite; (X) eliciting earlier and strong CT (CD8+T) response by stimulating intra hepatic memory lymphocytes upon Plasmodium infection; and (XI) preventing malaria parasite escape from immune response thereby allowing long-term control of the infection by the malaria parasite.

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